Toso as a target for drug screening

ABSTRACT

The present invention is directed to methods for identifying novel compositions which modulate the activity of Toso, and the use of such compositions in diagnosis and treatment of disease.

FIELD OF THE INVENTION

The invention relates to the use of Toso proteins in screening assays.

BACKGROUND OF THE INVENTION

Apoptosis or programmed cell death is an important homeostatic mechanismthat maintains cell number, positioning, and differentiation. Severalintracellular and intercellular processes are known to regulateapoptosis. One of the best characterized systems is initiated by thecell surface receptor, Fas (Apo-1/CD95), homologues of which initiateapoptosis in a wide range of organisms (Itoh, et al., Cell, 66:233-243(1991); Yonehara, et al., J. Exp. Med., 169:1747-1756 (1989)).Clustering of the Fas cytoplasmic domain generates an apoptotic signalvia the “death domain” (Itoh and Nagata, J. Biol. Chem., 268:10932-10937(1993)). Several critical proteins that bind to the death domain orother domains within the cytoplasmic region have been identified usingyeast two-hybrid and biochemical screens (Boldin, et al., J. Biol.Chem., 270:7795-7798 (1995); Chinnaiyan, et al., Cell, 8145:505-512(1995); Chu, et al., Proc. Natl. Acad. Sci. USA, 92:11894-11898 (1995);Okura, et al., J. Immunol., 157:4277-4281 (1996); Sato, et al., Science,268:411-415 (1995); Stanger, et al., Cell, 8145:513-523 (1995)).

Fas engagement by Fas ligand is capable of activating the interleukin-1β converting enzyme family of cysteine proteases (Caspases)—theproteolytic executors of apoptosis (Enari, et al., Nature, 375:78-81(1995); Enari, et al., Nature, 380:723-726 (1996); Los, et al., Nature,375:81-83 (1995); Tewari and Dixit, J. Biol. Chem, 270:3255-3260(1995)). Recent studies implicate caspase8 (MACH/FLICE/Mch5) as linkingFas receptor signaling to downstream caspases via its association withFADD/MORT1 (Boldin, et al., (1995); Chinnaiyan, et al., (1995); Boldin,et al., (1996); Fernandes-Alnemri, et al., Proc. Natl. Acad Sci. USA,93:7464-7469 (1996); Muzio, et al., Cell, 85:817-827 (1996)). Severalgroups have reported that caspase-8 activation is inhibited by acellular inhibitor, cFLIP/FLAME-1/1-FLICE (Irmler, et al., Nature,388:190-195 (1997); Srinivasula, et al., J. Biol. Chem., 272:18542-18545(1997); Hu, et al., J. Biol. Chem., 272:17255-17257 (1997)). Otherproteins involved in Fas-mediated apoptosis include: (a) theFas-activated serine/threonine kinase (FAST kinase), which is rapidlyactivated during Fas-mediated apoptosis; (b) acid sphingomyelinase,which produces ceramide, a pro-apoptotic signal that acts as a secondmessenger in several systems; and (c) Daxx, a novel protein that linksFas to the JNK stress kinase pathway (Cifone, et al., J. Exp. Med.,180:1547-1552 (1994); Tian, et al., J. Exp. Med., 182:865-874 (1995);Yang, et al., Cell, 89:1067-1076 (1997)). The exact role of these latterco-activators has yet to be fully defined.

A balance between life and programmed cell death signals in cells islikely to be governed by multiple interacting regulators that counteractapoptotic signals with appropriate anti-apoptotic signals. Imbalances inthis regulation can result in wide variety of pathologies, includingcancer and immune dysfunction and it is now clear that otherpolypeptides besides Fas contribute to disregulation of appropriatelyinduced apoptosis. As an example, in many tumor cell lines Fasexpression does not correlate with sensitivity to Fas-induced apoptosis,implying the existence of Fas-resistance protein (Richardson, et al.,Eur. J. Immunol., 24:2640-2645 (1994)). Also, in some types of cells,Fas-induced apoptosis requires protein synthesis inhibitors such ascycloheximide (Itoh and Nagata, (1993); Yonehara, et al., (1989)) andeven in Fas-sensitive cells, protein synthesis inhibitors can play asynergistic role with cycloheximide (Itoh and Nagata, (1993)). Thesecombined observations further suggest the existence of proteins capableof suppressing Fas-generated apoptotic signaling.

Additionally, in the course of a normal immune response, both cytotoxicT cell and NK cell activation can lead to Fas ligand (FasL) induction ofapoptosis in target cells (Arase, et al., J. Exp. Med., 181:1235-1238(1995); Berke, Cell, 81:9-12 (1995); Montel, et al., Cell Immunol.,166:236-246 (1995)). Although both Fas and FasL are rapidly inducedfollowing T-cell activation, activated-T cells remain resistant toFas-induced apoptosis for several days (Klas, et al., Int. Immunol.,5:625-630 (1993); Owen-Schaub, et al, Cell Immunol., 140:197-205(1992)). Thus, a mechanism exists to shield newly activated T cells fromthe cytotoxicity of their own FasL expression. This is likely to be animportant component of T cell activation processes and protection inlymph nodes, splenic germinal centers and other sites at which T cellactivation results in apoptosis of target cells.

Described herein is the identification and characterization of a novelsurface molecule, “Toso” which is a member of the immunoglobulin genesuperfamily and which specifically inhibits Fas and TNF receptor familymediated apoptosis. The results demonstrate the existence of cellsurface mediated signaling pathways that lead to down regulation ofFas-mediated apoptosis in certain cell types and suggest that activationof T cells suppresses internal signaling systems that might leadinappropriately to T cell-induced self-killing.

Accordingly, it is an object of the invention to provide Toso proteinsand related molecules. It is a further object of the invention toprovide recombinant nucleic acids encoding Toso proteins, and expressionvectors and host cells containing the nucleic acid encoding the Tosoprotein. A further object of the invention is to provide methods forscreening for antagonists and agonists of Toso.

SUMMARY OF THE INVENTION

In accordance with the objects outlined above, the present inventionprovides methods for screening for a bioactive agent capable of bindingto a Toso protein encoded by a recombinant nucleic acid that willhybridize under high stringency conditions to the nucleic acid sequencedepicted in FIG. 1 (SEQ ID NO:1) or its complement. The methods comprisecombining a Toso protein and a candidate bioactive agent, anddetermining the binding of the candidate agent to the Toso protein.

In an additional aspect, the invention provides methods for screeningfor a bioactive agent capable of modulating the activity of a Tosocell-surface receptor, said method comprising the steps of adding acandidate bioactive agent to a cell comprising a recombinant nucleicacid encoding a Toso receptor, exposing the cells to an apoptotic agentthat will induce apoptosis, and determining the effect of the candidatebioactive agent on apoptosis.

In a further aspect, the invention provides methods of modulatingapoptosis in a cell comprising administering to the cell an exogenouscompound that binds to a Toso protein wherein the binding modulates thebiological activity of said Toso protein.

In an additional aspect, the invention provides methods for identifyinga cell containing a mutant Toso gene comprising determining the sequenceof all or part of at least one of the endogenous Toso genes. Similarly,methods of identifying the Toso genotype of an individual are provided.

In a further aspect, the invention provides methods for diagnosing anapoptosis related condition in an individual. The activity of Toso in atissue from a first individual is measured and compared to the activityof Toso in a tissue from a second, unaffected individual or from asecond tissue in the first individual. When the activity of Toso fromsaid first individual is less than the activity of Toso in the secondindividual, the first individual is at risk for an apoptosis relatedcondition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence (SEQ ID:NO 1) of Toso. Alsopresented are the positions of the initiator ATG start codon, the stopcodon the nucleotides which correspond to the signal sequence and thenucleotides which correspond to the putative transmembrane domain of theToso protein.

FIG. 2a depicts the amino acid sequence (SEQ ID:NO 2) of amino acids 1to 390 deduced from nucleotides 1 to 1173 of the nucleotide sequenceshown in FIG. 1 (SEQ ID:NO 1). Two hydrophobic regions are underlined.

FIG. 2b depicts a Kyte-Dolittle hydropathy plot analysis of Toso geneproduct (upper) and schematic presentation of Toso (bottom). The matureToso is a 390-amino acid protein with the leader sequence of 17 aminoacids (hatched bar), the extracellular domain of 236 amino acids (ED),the transmembrane region of 19 amino acids (TM; dotted bar) and thecytoplasmic domain of 118 amino acids (CD). The immunoglobulin domain(Ig), the basic amino acid-rich region (Basic), the proline-rich region(Proline), and the acidic amino acid-rich region (Acidic) are indicated.

FIG. 3 (SEQ ID NOS:3-21) depicts BLAST search results using the Tosogene product. The position of the first amino acid in each sequence isgiven in the left side of the alignment. Gaps are indicated by dashes.Dark and light shading refer to identical and similar residues,respectively. For sequence alignment of the Toso N-terminus, IgVH(GlHUNM), IgVλ (L1MS4E), TcR Vα (RWMSAV), TCR Vβ (RWHUVY), CD4 (U47924),CD8 chain 11 (X04310), Poly Ig R (QRRBG) and immunoglobulin V-setconsensus sequence are shown in the alignment. Arrows indicate positionscharacteristic of many V-set sequences. The sequence of the Tosocytoplasmic domain is aligned with acid sphingomyelinase, insulinreceptor substrate 1 (IRS1) and apoptosis inhibitor, IAP, from Orgyiapseudotsugata nuclear polyhedrosis virus (Op-1AP).

FIG. 4a depicts the effect of Toso on anti-Fas induced apoptosis. Thepercentage of apoptotic cells are expressed as the mean (hatched andshaded bar)±SD of triplicate cultures. Apoptotic cells in each culturewithout anti-Fas mAb were less than 2%.

FIG. 4b depicts the effect of Toso on anti-Fas-, staurosporine- andceramide-induced apoptosis in Jurkat.ecoR cells (closed triangle),Jurkat.ecoR cells infected with pBabeMN-lacZ (closed square) andpBabeMN-Toso (open circle). The percentage of apoptotic cells areexpressed as the mean (symbol)±SD (vertical bar) of triplicate cultures.

FIG. 4c depicts the effect of Toso on FADD-induced apoptosis inJurkat.ecoR cells infected with pBabeMN-Lyt-2-α′ (hatched bar), andpBabeMN-Toso (shaded bar). The percentage of apoptotic cells areexpressed as the mean (hatched bar or shaded bar)±SD of triplicatecultures.

FIG. 4d depicts the effect of Toso on TNF-α-induced apoptosis inJurkat.ecoR cells. The percentage of apoptotic cells are expressed asthe mean (hatched bar or shaded bar)±SD of triplicate cultures.

FIG. 4e depicts the effect Toso on anti-Fas mAb-induced apoptosis incells cultured with (α-Fas (+)) or without (α-Fas (−)) 50 ng/ml ofanti-Fas mAb. After culture for five days, GFP expression of survivedcells were analyzed by FACScan.

FIG. 5a depicts the results of Western blot analysis of caspase-8processing by induction of cFLIP. Jurkat.ecoR cells (control) andpBabeMN-Toso-infected Jurkat.ecoR cells (Toso) were cultured with (+) orwithout (−) 50 ng/ml of anti-Fas mAb (α-Fas) for 6 hours. Positions ofpro-caspase-8 (Pro), the processed form (p20) and standard marker areindicated.

FIG. 5b depicts the results of RT-PCR of cFLIP expression in Jurkat.ecoRcells (control) and pBabeMN-Toso-infected Jurkat.ecoR cells (Toso).

FIG. 6a depicts the effect of Toso deletion mutant expression onanti-Fas mAb-induced apoptosis. Structure of the Toso deletion mutantsis shown at the left side of this panel. Full-length Toso is a 390-aminoacid protein with the leader sequence of 17 amino acids (hatched bar),the extracellular domain of 236 amino acids (ED), the transmembraneregion of 19 amino acids (TM; dark-shaded bar) and the cytoplasmicdomain of 118 amino acids (CD). The hemagglutinin (HA) tag is indicatedas a light shaded bar. The percentage of apoptotic cells is expressed asthe mean (hatched and shaded bar)±SD of triplicate cultures.

FIG. 6b depicts Western blot analysis of deletion mutants using anti-HAantibody. The molecular weight of major products from Toso.HA,TosoΔ(377-390).HA, TosoΔ (334-390). HA, TosoΔ(252-390). HA,TosoΔ(281-390). HA, TosoΔ(29 187). HA and Lyt-2/Toso(271-390).HA was60/35, 55/30, 50/26, 40, 38, 35, 60/30 kDa, respectively. Positions andsizes (kDa) of standard protein markers are indicated in left side ofpanel.

FIG. 6c depicts Crosslinking the extracellular domain of Toso. Positionsof standard protein markers and Toso.HA are indicated in left side andright of panel, respectively.

FIG. 7a depicts mRNA dot blot analysis of Toso gene in several humantissues.

FIG. 7b depicts Northern blot analysis of Toso gene in several humanimmune tissues. Positions and sizes (kbp) of Toso mRNA are indicated inleft side of panels.

FIG. 7c depicts RT-PCR analysis of Toso in human cell lines (upperpanel). Positions and sizes (kbp) of Toso and standard nucleotide makersare indicated. As a control for loading, we amplified β-actin cDNA(lower panels).

FIG. 8a depicts (a) Northern blot analysis of Toso gene in Jurkat cells(None) and Jurkat cells stimulated with PMA and PHA (PMA+PHA) or PMA andlonomycin (PMA+lo.). RNA was electrophoresed, transferred to a Hybond N+membrane and hybridized with a radiolabelled probe specific for Toso(upper) and β-actin (lower). Film was exposed at −70° C. with anintensifying screen for two days (upper). Positions and sizes (kbp) ofToso mRNA are indicated in right side of panels.

FIG. 8b depicts activation induced resistance to anti-Fas mAb-inducedapoptosis in Jurkat cells. The percentage of apoptotic cells areexpressed as the mean (hatched bar)±SD of triplicate cultures.

FIG. 8c depicts the effect of Toso on PMA and lonomycin(PMA+lo.)-induced apoptosis. Jurkat.ecoR cells (−), Jurkat.ecoR cellsinfected with pBabeMN-lacZ (lacZ), pBabeMN-Toso-infected clones (Tosoclones 1-5) were cultured with 10 ng/ml of anti-Fas mAb (left), 10 ng/mlPMA and 500 ng/ml lonomycin (right) for 24 hours. The percentage ofapoptotic cells are expressed as the mean (hatched bar and shadedbar)±SD of triplicate cultures.

FIG. 9a depicts the RT-PCR analysis of Toso in peripheral bloodmononuclear cells after activation with PHA (upper panel, the 1.2 kbpfragment of Toso).

FIG. 9b depicts analysis of Toso in peripheral blood mononuclear cellsafter allogenic stimulation (upper panel, the 1.2 kbp fragment of Toso).Stimulator cells (SC), responder cells (RC) or mixed cells (RC+SC) werecultured for one day (day 1) and six days (day 6).

FIG. 10 depicts a model for the role of Toso in T cell activation. Inthe model, the role of Toso is to be induced following T cell activationand to protect T cells from self-induced programmed cell death. Theinhibitory effects of Toso on Fas signaling maps at the level ofcaspase-8 through induced expression of cFLIP.

FIG. 11 depicts massive cell death of 70 Z3 cells induced by TOSO. 70Z/3cells were incubated with supernatant from ΦNX-E (closed triangle),viral supernatant of pBabeMN-Lyt-2α (closed square), or pBabeMN-TOSO(open circle) for 12 hours including the initial spinning at 2500 rpmfor 90 min. Infection frequency of pBabeMN-Lyt-2α was determined to be79% at 48 hours post infection. The percentage of viable cells atvarious time points are expressed as mean (symbol)±SD (vertical bar) oftriplicate cultures.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel Ig domain-containing Tosopolypeptides, with potent pathway-specific anti-apoptotic effects inhematopoietic cells. Toso (named after a Japanese liquor that is drunkon New Year's Day to celebrate long life and eternal youth) exerts aninhibitory activity against apoptosis induced by Fas-, TNF-α-, FADD andPMA/lonomycin but not against staurosporine- or ceramide-inducedapoptosis. Without being bound by theory, the mechanism of blockingapoptotic activation, and the pathway specificity of the effect, is mostlikely explained by Toso induction of cFLIP expression which inhibitscaspase-8 processing. Toso is expressed within lymphoid tissues andhematopoietic cells, and is enhanced after T-cell activation. Theseresults suggest that Toso plays an important role in the immune system.Surprisingly, Toso also displays pro-apoptotic effects; Toso promotedcell death in the murine preB cell line, 70Z/3 cells, an effect that isshown to be caused by the cytoplasmic domain.

Accordingly, the present invention provides Toso proteins and nucleicacids. In a preferred embodiment, the Toso proteins are from vertebratesand more preferably from mammals including dogs, cats and rabbits,rodents (including rats, mice, hamsters, guinea pigs, etc.), primates(including chimpanzees, African green monkeys, etc.), farm animals(including sheep, goats, pigs, cows, horses, etc.) and in the mostpreferred embodiment, from humans. However, using the techniquesoutlined below, Toso proteins from other organisms may also be obtained.

As outlined herein, the Toso proteins of the present invention are Igsuperfamily molecules which are expressed in a variety of tissue types,including, but not limited to lymph nodes, peripheral blood leukocytes,thymus, lung, and kidney. As further outlined herein, Toso proteinsexert pathway specific anti-apoptotic effects in hematopoietic cells.Toso is a membrane bound protein, as it contains a putativetransmembrane domain. The extracellular domain of Toso has homology toimmunoglobulin variable domains.

A Toso protein of the present invention may be identified in severalways. “Protein” in this sense includes proteins, polypeptides, andpeptides. A Toso nucleic acid or Toso protein is initially identified bysubstantial nucleic acid and/or amino acid sequence homology to thesequences shown in FIGS. 1 and 2a. Such homology can be based upon theoverall nucleic acid or amino acid sequence.

As used herein, a protein is a “Toso protein” if the overall homology ofthe protein sequence to the amino acid sequence shown in FIG. 2a (SEQ IDNO:2) is preferably greater than about 50 or 60%, more preferablygreater than about 70 or 75%, even more preferably greater than about80% and most preferably greater than 85%. In some embodiments thehomology will be as high as about 90 to 95 or 98%. Homology in thiscontext means sequence similarity or identity, with identity beingpreferred. Identical in this context means identical amino acids atcorresponding positions in the two sequences which are being compared.Homology in this context includes amino acids which are identical andthose which are similar (functionally equivalent). This homology will bedetermined using standard techniques known in the art, such as the BestFit sequence program described by Devereux, et al., Nucl. Acid Res.,12:387-395 (1984), preferably using the default settings, or the BLASTXprogram (Altschul, et al., J. Mol. Biol., 215:403-410 (1990)). Thealignment may include the introduction of gaps in the sequences to bealigned. In addition, for sequences which contain either more or feweramino acids than the proteins shown in the Figures, it is understoodthat the percentage of homology will be determined based on the numberof homologous amino acids in relation to the total number of aminoacids. Thus, for example, homology of sequences shorter than that shownin the Figures, as discussed below, will be determined using the numberof amino acids in the shorter sequence.

As outlined herein, Toso proteins have several important domains. Tosocontains a cytoplasmic domain from amino acids 273 to 390, with theextracellular domain spanning from 18 to 253 (unless otherwisespecified, all amino acid numbering is based on the human sequence).Toso contains a standard transmembrane domain, spanning from amino acids254 to 272. Toso contains an additional hydrophobic region at theN-terminus, amino acids 1 to 17, corresponding to a putative signalsequence. In addition, the cytoplasmic domain of Toso contains a basicamino acid-rich region (from Arg274 to Arg323), a proline rich region(from Pro334 to P346), and an acidic amino acid-rich region (from Glu378to Asp384). In addition, the cytoplasmic domain has partial homology toFAST kinase, acid sphingomyleinase, insulin receptor substrate-1 (IRS-1)and the apoptosis inhibitor from Orgyia pseudotsugata nuclearpolyhedrosis virus (Op-1AP). The extracellular domain of Toso hashomology to the immunoglobulin V-region.

As used herein, a protein is also a “Toso protein” if the homology ofthe cytoplasmic domain comprising amino acids 273 to 390, or theextracellular domain comprising amino acids 18 to 253, respectively, ofthe amino acid sequence shown in FIG. 2a (SEQ ID NO:2) is preferablygreater than about 50% of 60%, more preferably greater than about 70% or75%, even more preferably greater than about 80% and most preferablygreater than 85%. In some embodiments the homology will be as high asabout 90 to 95 or 98%.

Toso proteins of the present invention may be shorter or longer than theamino acid sequences shown in the Figures. Thus, in a preferredembodiment, included within the definition of Toso proteins are portionsor fragments of the sequences depicted in the Figures. As outlinedherein, Toso deletion mutants can be made, including, but not limitedto, the deletion of amino acids 377-390, 334-390, 281-390, 252-390, and29-187. As further outlined herein, Toso fusion proteins can be madeincluding, but not limited to, the fusion of amino acids 1-271. Apreferred Toso fragment is the cytoplasmic domain of Toso, which maymodulate apoptosis, as shown herein. A further preferred Toso fragmentis the extracellular domain of Toso, comprising roughly the first 236amino acids of Toso, which is required for the anti-apoptotic effects onanti-Fas antibody-stimulated cells. However, as outlined herein,preferred fragments of Toso also include a transmembrane domain, as itmay be involved in signaling and Fas-induced apoptosis by Toso mayrequire its insertion into membranes.

Thus, in a preferred embodiment, the Toso proteins of the presentinvention are Toso polypeptides. In this embodiment, a Toso polypeptidecomprises at least the immunoglobin V-like domain, and preferably atransmembrane domain, although it may contain additional amino acids aswell. As shown in the Examples and discussed below, Toso is an Igsuperfamily protein which is capable of inhibiting apoptosis mediated bymembers of the Fas or TNF receptor family of proteins.

In a preferred embodiment, the Toso proteins are derivative or variantToso proteins. That is, as outlined more fully below, the derivativeToso peptide will contain at least one amino acid substitution, deletionor insertion, with amino acid substitutions being particularlypreferred. The amino acid substitution, insertion or deletion may occurat any residue within the Toso peptide. As outlined below, particularlypreferred substitutions are made within the extracellular domain orcytoplasmic domain of the Toso protein.

In addition, as is more fully outlined below, Toso proteins can be madethat are longer than those depicted in the figures, for example, by theaddition of epitope or purification tags, the addition of other fusionsequences, etc.

Toso proteins may also be identified as being encoded by Toso nucleicacids. Thus, Toso proteins are encoded by nucleic acids that willhybridize to the sequence depicted in FIG. 1 (SEQ ID NO:1) or itscomplement, as outlined herein.

In a preferred embodiment, when the Toso protein is to be used togenerate antibodies, the Toso protein must share at least one epitope ordeterminant with the full length protein shown in FIG. 2a (SEQ ID NO:2).By “epitope” or “determinant” herein is meant a portion of a proteinwhich will generate and/or bind an antibody. Thus, in most instances,antibodies made to a smaller Toso protein will be able to bind to thefull length protein. In a preferred embodiment, the epitope is unique;that is, antibodies generated to a unique epitope show little or nocross-reactivity. In a preferred embodiment, the antibodies aregenerated to an extracellular portion of the Toso molecule, i.e. to allor some of the N-terminal region from amino acid numbers 18-253.

In a preferred embodiment, the antibodies to Toso are capable ofreducing or eliminating the biological function of Toso, as is describedbelow. That is, the addition of anti-Toso antibodies (either polyclonalor preferably monoclonal) to cells comprising Toso receptors may reduceor eliminate the Toso receptor activity, blocking the signaling pathwaythat blocks apoptosis; that is, when Toso receptor function is reducedor eliminated, the cells die. Generally, at least a 50% decrease inactivity is preferred, with at least about 75% being particularlypreferred and about a 95-100% decrease being especially preferred.

The Toso antibodies of the invention specifically bind to Toso proteins.By “specifically bind” herein is meant that the antibodies bind to theprotein with a binding constant in the range of at least 10⁶-10⁸ M, witha preferred range being 10⁷-9 M.

In the case of the nucleic acid, the overall homology of the nucleicacid sequence is commensurate with amino acid homology but takes intoaccount the degeneracy in the genetic code and codon bias of differentorganisms. Accordingly, the nucleic acid sequence homology may be eitherlower or higher than that of the protein sequence. Thus the homology ofthe nucleic acid sequence as compared to the nucleic acid sequence ofFIG. 1 (SEQ ID NO:1) is preferably greater than 50 or 60%, morepreferably greater than about 70 to 75%, particularly greater than about80% and most preferably greater than 85%. In some embodiments thehomology will be as high as about 90 to 95 or 98%.

In a preferred embodiment, a Toso nucleic acid encodes a Toso protein.As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the Toso proteins of the present invention. Thus,having identified a particular amino acid sequence, those skilled in theart could make any number of different nucleic acids, by simplymodifying the sequence of one or more codons in a way which does notchange the amino acid sequence of the Toso.

In one embodiment, the nucleic acid homology is determined throughhybridization studies. Thus, for example, nucleic acids which hybridizeunder high stringency to the nucleic acid sequences shown in FIG. 1 (SEQID NO:1) or its complement is considered a Toso gene. High stringencyconditions are known in the art; see for example Maniatis, et al.,Molecular Cloning: A Laboratory Manual, 2d Edition (1989), and ShortProtocols in Molecular Biology, ed. Ausubel, et al., both of which arehereby incorporated by reference. An example of such conditions includeshybridization at about 42° C. in about 6×SSC with 50% formamide andwashing conditions of about 65° C. in about 0.2×SSC, 0.1×SDS.

In another embodiment, less stringent hybridization conditions are used;for example, moderate or low stringency conditions may be used, as areknown in the art; see Maniatis and Ausubel, supra. An example of suchconditions includes hybridization at about 50 to 55° C. in 5×SSPE andwashing conditions of about 50° C. in about 5×SSPE.

The Toso proteins and nucleic acids of the present invention arepreferably recombinant. As used herein, “nucleic acid” may refer toeither DNA or RNA, or molecules which contain both deoxy- andribonucleotides. The nucleic acids include genomic DNA, cDNA andoligonucleotides including sense and anti-sense nucleic acids. Suchnucleic acids may also contain modifications in the ribose-phosphatebackbone to increase stability and half life of such molecules inphysiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequence depicted in FIG. 1 also includes the complement of thesequence. By the term “recombinant nucleic acid” herein is meant nucleicacid, originally formed in vitro, in general, by the manipulation ofnucleic acid by endonucleases, in a form not normally found in nature.Thus an isolated Toso nucleic acid, in a linear form, or an expressionvector formed in vitro by ligating DNA molecules that are not normallyjoined, are both considered recombinant for the purposes of thisinvention. It is understood that once a recombinant nucleic acid is madeand reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e. using the in vivo cellular machinery of the hostcell rather than in vitro manipulations; however, such nucleic acids,once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention.

Similarly, a “recombinant protein” is a protein made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdepicted above. A recombinant protein is distinguished from naturallyoccurring protein by at least one or more characteristics. For example,the protein may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedprotein is unaccompanied by at least some of the material with which itis normally associated in its natural state, preferably constituting atleast about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure protein comprisesat least about 75% by weight of the total protein, with at least about80% being preferred, and at least about 90% being particularlypreferred. The definition includes the production of a Toso protein fromone organism in a different organism or host cell. Alternatively, theprotein may be made at a significantly higher concentration than isnormally seen, through the use of a inducible promoter or highexpression promoter, such that the protein is made at increasedconcentration levels. Alternatively, the protein may be in a form notnormally found in nature, as in the addition of an epitope tag or aminoacid substitutions, insertions and deletions, as discussed below.

Once identified, the polypeptides comprising the biologically activesequences may be prepared in accordance with conventional techniques,such as synthesis (for example, use of a Beckman Model 990 peptidesynthesizer or other commercial synthesizer).

Also included within the definition of Toso proteins of the presentinvention are amino acid sequence variants. These variants fall into oneor more of three classes: substitutional, insertional or deletionalvariants. These variants ordinarily are prepared by site specificmutagenesis of nucleotides in the DNA encoding the Toso protein, usingcassette or PCR mutagenesis or other techniques well known in the art,to produce DNA encoding the variant, and thereafter expressing the DNAin recombinant cell culture as outlined above. However, variant Tosoprotein fragments having up to about 100-150 residues may be prepared byin vitro synthesis using established techniques. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the Toso protein amino acid sequence. Thevariants typically exhibit the same qualitative biological activity asthe naturally occurring analogue, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not to bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed Toso variants screened for theoptimal combination of desired activity. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known, for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants is done using assays of Tosoprotein activities; for example, for binding domain mutations,competitive binding studies such as are outlined in the Examples may bedone.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger. For example, a preferred variant comprises the deletion ofthe cytoplasmic domain, leaving only the extracellular domain of Toso,preferably including the transmembrane domain. Additional preferredvariants comprise the cytoplasmic domain alone or a soluble receptor (ie. the extracellular domain lacking the transmembrane domain).

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the Toso protein aredesired, substitutions are generally made in accordance with thefollowing chart:

Chart I Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g., lysyl, arginyl, or histidyl, is substituted for (orby) an electronegative residue, e.g., glutamyl or aspartyl; or (d) aresidue having a bulky side chain, e.g., phenylalanine, is substitutedfor (or by) one not having a side chain, e.g., glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the Toso proteins as needed. Alternatively, thevariant may be designed such that the biological activity of the Tosoprotein is altered. For example, glycosylation sites, and moreparticularly one or more O-linked or N-linked gylcosylation sites may bealtered or removed. Either or both of the transmembrane domains may bealtered or removed, to make a soluble or secreted protein, i.e. theextracellular domain.

Covalent modifications of Toso polypeptides are included within thescope of this invention. One type of covalent modification includesreacting targeted amino acid residues of a Toso polypeptide with anorganic derivatizing agent that is capable of reacting with selectedside chains or the N-or C-terminal residues of a Toso polypeptide.Derivatization with bifunctional agents is useful, for instance, forcrosslinking Toso to a water-insoluble support matrix or surface for usein the method for purifying anti-Toso antibodies or screening assays, asis more fully described below. Commonly used crosslinking agentsinclude, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis-(succinimidylpropionate), bifunctional maleimides suchas bis-N-maleimido-1,8-octane and agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginylresidues to the corresponding glutamyl and aspartyl residues,respectively, hydroxylation of proline and lysine, phosphorylation ofhydroxyl groups of seryl or threonyl residues, methylation of the“-amino groups of lysine, arginine, and histidine side chains [T. E.Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman &Co., San Francisco, pp. 79-86 (1983)], acetylation of the N-terminalamine, and amidation of any C-terninal carboxyl group.

Another type of covalent modification of the Toso polypeptide includedwithin the scope of this invention comprises altering the nativeglycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence Tosopolypeptide, and/or adding one or more glycosylation sites that are notpresent in the native sequence Toso polypeptide.

Addition of glycosylation sites to Toso polypeptides may be accomplishedby altering the amino acid sequence thereof. The alteration may be made,for example, by the addition of, or substitution by, one or more serineor threonine residues to the native sequence Toso polypeptide (forO-linked glycosylation sites). The Toso amino acid sequence mayoptionally be altered through changes at the DNA level, particularly bymutating the DNA encoding the Toso polypeptide at preselected bases suchthat codons are generated that will translate into the desired aminoacids.

Another means of increasing the number of carbohydrate moieties on theToso polypeptide is by chemical or enzymatic coupling of glycosides tothe polypeptide. Such methods are described in the art, e.g., in WO87/05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the Toso polypeptide may beaccomplished chemically or enzymatically or by mutational substitutionof codons encoding for amino acid residues that serve as targets forglycosylation. Chemical deglycosylation techniques are known in the artand described, for instance, by Hakimuddin, et al., Arch. Biochem.Biophys., 259:52 (1987) and by Edge, et al., Anal. Biochem., 118:131(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides canbe achieved by the use of a variety of endo-and exo-glycosidases asdescribed by Thotakura, et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of Toso comprises linking the Tosopolypeptide to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in themanner set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;4,670,417; 4,791,192; or 4,179,337.

Toso polypeptides of the present invention may also be modified in a wayto form chimeric molecules comprising a Toso polypeptide fused toanother, heterologous polypeptide or amino acid sequence. In oneembodiment, such a chimeric molecule comprises a fusion of a Tosopolypeptide with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino-or carboxyl-terminus of the Toso polypeptide. Thepresence of such epitope-tagged forms of a Toso polypeptide can bedetected using an antibody against the tag polypeptide. Also, provisionof the epitope tag enables the Toso polypeptide to be readily purifiedby affinity purification using an anti-tag antibody or another type ofaffinity matrix that binds to the epitope tag. In an alternativeembodiment, the chimeric molecule may comprise a fusion of a Tosopolypeptide with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule, such afusion could be to the Fc region of an IgG molecule or GST fusions.

Various tag polypeptides and their respective antibodies are well knownin the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field, et al., Mol. Cell Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7, and 9E10antibodies thereto [Evan, et al., Molecular and Cellular Biology,5:3610-3616(1985)]; and the Herpes Simplex virus glycoprotein D (gD) tagand its antibody [Paborsky, et al., Protein Engineering, 3(6):547-553(1990)]. Other tag polypeptides include the Flag-peptide [Hopp, et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin, etal., Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner, etal., J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 proteinpeptide tag [Lutz-Freyermuth, et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

Also included with the definition of Toso protein are other Tosoproteins of the Toso family, and Toso proteins from other organisms,which are cloned and expressed as outlined below. Thus, probe ordegenerate polymerase chain reaction (PCR) primer sequences may be usedto find other related Toso proteins from humans or other organisms. Aswill be appreciated by those in the art, particularly useful probeand/or PCR primer sequences include the unique areas of the Toso nucleicacid sequence. Thus, useful probe or primer sequences may be designedto: all or part of the sequence of the immunoglobulin V-like Tosodomain, all or part of the unique extracellular domain, which spansroughly amino acids 18-253, or sequences outside the coding region. Asis generally known in the art, preferred PCR primers are from about 15to about 35 nucleotides in length, with from about 20 to about 30 beingpreferred, and may contain inosine as needed. The conditions for the PCRreaction are well known in the art.

Once the Toso nucleic acid is identified, it can be cloned and, ifnecessary, its constituent parts recombined to form the entire Tosonucleic acid. Once isolated from its natural source, e.g., containedwithin a plasmid or other vector or excised therefrom as a linearnucleic acid segment, the recombinant Toso nucleic acid can befurther-used as a probe to identify and isolate other Toso nucleicacids. It can also be used as a “precursor” nucleic acid to makemodified or variant Toso nucleic acids and proteins.

Using the nucleic acids of the present invention which encode a Tosoprotein, a variety of expression vectors are made. The expressionvectors may be either self-replicating extrachromosomal vectors orvectors which integrate into a host genome. Generally, these expressionvectors include transcriptional and translational regulatory nucleicacid operably linked to the nucleic acid encoding the Toso protein. Theterm “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the Toso protein; for example, transcriptional andtranslational regulatory nucleic acid sequences from Bacillus arepreferably used to express the Toso protein in Bacillus. Numerous typesof appropriate expression vectors, and suitable regulatory sequences areknown in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

A preferred expression vector system is a retroviral vector system suchas is generally described in PCT/US97/01019 and PCT/US97/01048, both ofwhich are hereby expressly incorporated by reference.

The Toso proteins of the present invention are produced by culturing ahost cell transformed with an expression vector containing nucleic acidencoding a Toso protein, under the appropriate conditions to induce orcause expression of the Toso protein. The conditions appropriate forToso protein expression will vary with the choice of the expressionvector and the host cell, and will be easily ascertained by one skilledin the art through routine experimentation. For example, the use ofconstitutive promoters in the expression vector will require optimizingthe growth and proliferation of the host cell, while the use of aninducible promoter requires the appropriate growth conditions forinduction. In addition, in some embodiments, the timing of the harvestis important. For example, the baculoviral systems used in insect cellexpression are lytic viruses, and thus harvest time selection can becrucial for product yield.

Appropriate host cells include yeast, bacteria, archebacteria, fungi,and insect and animal cells, including mammalian cells, for exampleprimary cells, including stem cells, including, but not limited to bonemarrow stem cells. Of particular interest are Drosophila melangastercells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillussubtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS,and HeLa cells, fibroblasts, Schwanoma cell lines, immortalizedmammalian myeloid and lymphoid cell lines, Jukat cells, human cells andother primary cells.

In a preferred embodiment, the Toso proteins are expressed in mammaliancells. Mammalian expression systems are also known in the art, andinclude retroviral systems. A mammalian promoter is any DNA sequencecapable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for Toso protein intomRNA. A promoter will have a transcription initiating region, which isusually placed proximal to the 5′ end of the coding sequence, and a TATAbox, using a located 25-30 base pairs upstream of the transcriptioninitiation site. The TATA box is thought to direct RNA polymerase II tobegin RNA synthesis at the correct site. A mammalian promoter will alsocontain an upstream promoter element (enhancer element), typicallylocated within 100 to 200 base pairs upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation. Of particular use asmammalian promoters are the promoters from mammalian viral genes, sincethe viral genes are often highly expressed and have a broad host range.Examples include the SV40 early promoter, mouse mammary tumor virus LTRpromoter, adenovirus major late promoter, herpes simplex virus promoter,the CMV promoter, a retroviral LTR promoter, mouse maloney luekemiavirus LTR, or pBabeMN.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenlytion signals includethose derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

In a preferred embodiment, Toso proteins are expressed in bacterialsystems. Bacterial expression systems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3′)transcription of the coding sequence of Toso protein into mRNA. Abacterial promoter has a transcription initiation region which isusually placed proximal to the 5′ end of the coding sequence. Thistranscription initiation region typically includes an RNA polymerasebinding site and a transcription initiation site. Sequences encodingmetabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non- bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. coli, the ribosome binding site iscalled the Shine-Delgamo (SD) sequence and includes an initiation codonand a sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon.

The expression vector may also include a signal peptide sequence thatprovides for secretion of the Toso protein in bacteria. The signalsequence typically encodes a signal peptide comprised of hydrophobicamino acids which direct the secretion of the protein from the cell, asis well known in the art. The protein is either secreted into the growthmedia (gram-positive bacteria) or into the periplasmic space, locatedbetween the inner and outer membrane of the cell (gram-negativebacteria).

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterial hostcells using techniques well known in the art, such as calcium chloridetreatment, electroporation, and others.

In one embodiment, Toso proteins are produced in insect cells.Expression vectors for the transformation of insect cells, and inparticular, baculovirus-based expression vectors, are well known in theart.

In a preferred embodiment, Toso protein is produced in yeast cells.Yeast expression systems are well known in the art, and includeexpression vectors for Saccharomyces cerevisiae, Candida albicans and C.maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis,Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, andYarrowia lipolytica. Preferred promoter sequences for expression inyeast include the inducible GAL1,10 promoter, the promoters from alcoholdehydrogenase, enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUPI gene, which allows yeast to grow in the presence of copperions.

The Toso protein may also be made as a fusion protein, using techniqueswell known in the art. Thus, for example, for the creation of monoclonalantibodies, if the desired epitope is small, the Toso protein may befused to a carrier protein to form an immunogen. Alternatively, the Tosoprotein may be made as a fusion protein to increase expression, or forother reasons. For example, when the Toso protein is a Toso peptide, thenucleic acid encoding the peptide may be linked to other nucleic acidfor expression purposes.

In one embodiment, the Toso nucleic acids, proteins and antibodies ofthe invention are labeled. By “labeled” herein is meant that a compoundhas at least one element, isotope or chemical compound attached toenable the detection of the compound. In general, labels fall into threeclasses: a) isotopic labels, which may be radioactive or heavy isotopes;b) immune labels, which may be antibodies or antigens; and c) colored orfluorescent dyes. The labels may be incorporated into the compound atany position.

In a preferred embodiment, the Toso protein is purified or isolatedafter expression. Toso proteins may be isolated or purified in a varietyof ways known to those skilled in the art depending on what othercomponents are present in the sample. Standard purification methodsinclude electrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the Toso protein may be purified using a standard anti-Toso antibodycolumn. Ultrafiltration and diafiltration techniques, in conjunctionwith protein concentration, are also useful. For general guidance insuitable purification techniques, see Scopes, R., Protein Purification,Springer-Verlag, N.Y. (1982). The degree of purification necessary willvary depending on the use of the Toso protein. In some instances nopurification will be necessary.

Once expressed and purified if necessary, the Toso proteins and nucleicacids are useful in a number of applications.

In a preferred embodiment, modified Toso cell-surface receptors, andcells containing the modified receptors, are made. In one embodiment,non-human animals (preferably transgenic) are made that contain modifiedToso receptors; similarly, “knock-out” animal models and Toso transgenicanimals that contain an inducible promoter may be made.

In a preferred embodiment, the Toso proteins, nucleic acids, modifiedreceptors and cells containing the native or modified receptors are usedin screening assays. Identification of this important receptor permitsthe design of drug screening assays for compounds that modulate Tosoreceptor activity.

Screens may be designed to first find candidate agents that can bind toToso receptors, and then these agents may be used in assays thatevaluate the ability of the candidate agent to modulate Toso activity.Thus, as will be appreciated by those in the art, there are a number ofdifferent assays which may be run; binding assays and activity assays.Of particular importance in these embodiments is that the extracellularportion of Toso is mainly responsible for the anti-apoptotic effects.Accordingly, candidate agents may be added directly to cells without theneed to target the agents intracellularly when assaying foranti-apoptotic effects. Of firther importance is that the cytoplasmicdomain of Toso has been shown to enhance apoptosis. Accordingly, boththe extracellular and cytoplasmic domains of Toso may be usedindependently as a basis for binding assays.

Thus, in a preferred embodiment, the methods comprise combining a Tosocell surface receptor and a candidate bioactive agent, and determiningthe binding of the candidate agent to the Toso receptor. Preferredembodiments utilize the human Toso cell surface receptor (or portions,as outlined herein, such as the extracellular domain or the cytoplasmicdomain), although other mammalian receptors may also be used in eithercase, including rodents (mice, rats, hamsters, guinea pigs, etc.), farmanimals (cows, sheep, pigs, horses, etc.) and primates. These latterembodiments may be preferred in the development of animal models ofhuman disease. In some embodiments, as outlined herein, variant orderivative Toso receptors may be used, including deletion Toso receptorsas outlined above.

Furthermore, included within the definition of Toso cell surfacereceptors are portions of Toso cell surface receptors; that is, eitherthe full-length receptor may be used, or functional portions thereof. Ina preferred embodiment, the extracellular domain of Toso may be usedwithout or without the transmembrane region. In an additional preferredembodiment, the cytoplasmic domain of Toso may be used without orwithout the transmembrane region. In addition, the assays describedherein may utilize either isolated Toso receptors (including bothsoluble and membrane or lipid bound receptors) or cells comprising theToso receptors, with the latter being preferred.

Generally, in a preferred embodiment of the methods herein, the Tosocell surface receptor or the candidate agent is non-diffusably bound toan insoluble support having isolated sample receiving areas (e.g., amicrotiter plate, an array, etc.). The insoluble supports may be made ofany composition to which the compositions can be bound, is readilyseparated from soluble material, and is otherwise compatible with theoverall method of screening. The surface of such supports may be solidor porous and of any convenient shape. Examples of suitable insolublesupports include microtiter plates, arrays, membranes and beads. Theseare typically made of glass, plastic (e.g., polystyrene),polysaccharides, nylon or nitrocellulose, Teflon™, etc. Microtiterplates and arrays are especially convenient because a large number ofassays can be carried out simultaneously, using small amounts ofreagents and samples. The particular manner of binding of thecomposition is not crucial so long as it is compatible with the reagentsand overall methods of the invention, maintains the activity of thecomposition and is nondiffusable. Preferred methods of binding includethe use of antibodies (which do not sterically block theapoptosis-modulating sequence when the Toso protein is bound to thesupport), direct binding to “sticky” or ionic supports, chemicalcrosslinking, the synthesis of the Toso protein or receptor on thesurface, etc. Following binding of the Toso protein or receptor, excessunbound material is removed by washing. The sample receiving areas maythen be blocked through incubation with bovine serum albumin (BSA),casein or other innocuous protein or other moiety.

A candidate bioactive agent is added to the assay. Novel binding agentsinclude specific antibodies, non-natural binding agents identified inscreens of chemical libraries, peptide analogs, etc. Of particularinterest are screening assays for agents that have a low toxicity forhuman cells. A wide variety of assays may be used for this purpose,including labeled in vitro protein-protein binding assays,electrophoretic mobility shift assays, immunoassays for protein binding,functional assays (phosphorylation assays, etc.) and the like.

The term “candidate bioactive agent” or “exogeneous compound” as usedherein describes any molecule, e.g., protein, oligopeptide, smallorganic molecule, polysaccharide, polynucleotide, etc., with thecapability of directly or indirectly modulating apoptosis, which can bein response to ligand binding or in the absence of ligand binding.Generally a plurality of assay mixtures are run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e., at zero concentration or below the level ofdetection.

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides. Alternatively, libraries of natural compounds in theform of bacterial, fungal, plant and animal extracts are available orreadily produced. Additionally, natural or synthetically producedlibraries and compounds are readily modified through conventionalchemical, physical and biochemical means. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification to producestructural analogs.

In a preferred embodiment, the candidate bioactive agents are proteins.By “protein” herein is meant at least two covalently attached aminoacids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations.

In a preferred embodiment, the candidate bioactive agents are naturallyoccuring proteins or fragments of naturally occuring proteins. Thus, forexample, cellular extracts containing proteins, or random or directeddigests of proteinaceous cellular extracts, may be used. In this waylibraries of procaryotic and eucaryotic proteins may be made forscreening against Toso. Particularly preferred in this embodiment arelibraries of bacterial, fungal, viral, and mammalian proteins, with thelatter being preferred, and human proteins being especially preferred.

In a preferred embodiment, the candidate bioactive agents are peptidesof from about 5 to about 30 amino acids, with from about 5 to about 20amino acids being preferred, and from about 7 to about 15 beingparticularly preferred. The peptides may be digests of naturallyoccuring proteins as is outlined above, random peptides, or “biased”random peptides. By “randomized” or grammatical equivalents herein ismeant that each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. Since generally these randompeptides (or nucleic acids, discussed below) are chemically synthesized,they may incorporate any nucleotide or amino acid at any position. Thesynthetic process can be designed to generate randomized proteins ornucleic acids, to allow the formation of all or most of the possiblecombinations over the length of the sequence, thus forming a library ofrandomized candidate bioactive proteinaceous agents.

In one embodiment, the library is fully randomized, with no sequencepreferences or constants at any position. In a preferred embodiment, thelibrary is biased. That is, some positions within the sequence areeither held constant, or are selected from a limited number ofpossibilities. For example, in a preferred embodiment, the nucleotidesor amino acid residues are randomized within a defined class, forexample, of hydrophobic amino acids, hydrophilic residues, stericallybiased (either small or large) residues, towards the creation ofcysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

In a preferred embodiment, the candidate bioactive agents are nucleicacids. By “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein means at least two nucleotides covalently linked together. Anucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925(1993) and references therein; Letsinger, J. Org. Chem., 35:3800 (1970);Sprinzl, et al., Eur. J. Biochem., 81:579 (1977); Letsinger, et al.,Nucl. Acids Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805(1984), Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); andPauwels, et al., Chemica Scripta, 26:141 (1986)), phosphorothioate (Mag,et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No.5,644,048), phosphorodithioate (Briu, et al., J. Am. Chem. Soc.,111:2321 (1989)), O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc., 114:1895 (1992); Meier, et al., Chem. Int. Ed. Engl.,31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson, et al.,Nature, 380:207 (1996), all of which are incorporated by reference)).Other analog nucleic acids include those with positive backbones(Denpcy, et al., Proc. Natl. Acad. Sci. USA, 92:6097 (1995)); non-ionicbackbones (U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141;and 4,469,863; Kiedrowshi, et al., Angew. Chem. Intl. Ed. English,30:423 (1991); Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988);Letsinger, et al., Nucleoside & Nucleotide, 13:1597 (1994); Chapters 2and 3, ASC Symposium Series 580, “Carbohydrate Modifications inAntisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, etal., Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et al., J.Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins,et al., Chem. Soc. Rev., (1995) pp. 169-176). Several nucleic acidanalogs are described in Rawls, C & E News, Jun. 2, 1997, page 35. Allof these references are hereby expressly incorporated by reference.These modifications of the ribose-phosphate backbone may be done tofacilitate the addition of additional moieties such as labels, or toincrease the stability and half-life of such molecules in physiologicalenvironments. In addition, mixtures of naturally occurring nucleic acidsand analogs can be made. Alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occuring nucleic acids andanalogs may be made. The nucleic acids may be single stranded or doublestranded, as specified, or contain portions of both double stranded orsingle stranded sequence. The nucleic acid may be DNA, both genomic andcDNA, RNA or a hybrid, where the nucleic acid contains any combinationof deoxyribo- and ribo-nucleotides, and any combination of bases,including uracil, adenine, thymine, cytosine, guanine, inosine,xathanine hypoxathanine, isocytosine, isoguanine, etc.

As described above generally for proteins, nucleic acid candidatebioactive agents may be naturally occuring nucleic acids, random nucleicacids, or “biased” random nucleic acids. For example, digests ofprocaryotic or eucaryotic genomes may be used as is outlined above forproteins.

In a preferred embodiment, the candidate bioactive agents are organicchemical moieties, a wide variety of which are available in theliterature.

The determination of the binding of the candidate bioactive agent to theToso receptor may be done in a number of ways. In a preferredembodiment, the candidate bioactive agent is labelled, and bindingdetermined directly. For example, this may be done by attaching all or aportion of the Toso cell-surface receptor to a solid support, adding alabelled candidate agent (for example a fluorescent label), washing offexcess reagent, and determining whether the label is present on thesolid support. Various blocking and washing steps may be utilized as isknown in the art.

By “labeled” herein is meant that the compound is either directly orindirectly labeled with a label which provides a detectable signal,e.g., radioisotope, fluorescers, enzyme, antibodies, particles such asmagnetic particles, chemiluminescers, or specific binding molecules,etc. Specific binding molecules include pairs, such as biotin andstreptavidin, digoxin and antidigoxin, etc. For the specific bindingmembers, the complementary member would normally be labeled with amolecule which provides for detection, in accordance with knownprocedures, as outlined above. The label can directly or indirectlyprovide a detectable signal.

In some embodiments, only one of the components is labeled. For example,the receptors (or proteinaceous candidate agents) may be labeled attyrosine positions using 125I, or with fluorophores. Alternatively, morethan one component may be labeled with different labels; using ¹²⁵I forthe receptors, for example, and a fluorophor for the candidate agents.

In a preferred embodiment, the binding of the candidate bioactive agentis determined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget molecule, such as an antibody, peptide, binding partner, ligand,etc. Under certain circumstances, there may be competitive binding asbetween the bioactive agent and the binding moiety, with the bindingmoiety displacing the bioactive agent.

In one embodiment, the candidate bioactive agent is labeled. Either thecandidate bioactive agent, or the competitor, or both, is added first tothe receptor for a time sufficient to allow binding, if present.Incubations may be performed at any temperature which facilitatesoptimal activity, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high through put screening. Typically between 0.1 and 1 hour willbe sufficient. Excess reagent is generally removed or washed away. Thesecond component is then added, and the presence or absence of thelabeled component is followed, to indicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate bioactive agent. Displacement of the competitor is anindication that the candidate bioactive agent is binding to the Tosoreceptor and thus is capable of binding to, and potentially modulating,the activity of the Toso receptor. In this embodiment, either componentcan be labeled. Thus, for example, if the competitor is labeled, thepresence of label in the wash solution indicates displacement by theagent. Alternatively, if the candidate bioactive agent is labeled, thepresence of the label on the support indicates displacement.

In an alternative embodiment, the candidate bioactive agent is addedfirst, with incubation and washing, followed by the competitor. Theabsence of binding by the competitor may indicate that the bioactiveagent is bound to the Toso receptor with a higher affinity. Thus, if thecandidate bioactive agent is labeled, the presence of the label on thesupport, coupled with a lack of competitor binding, may indicate thatthe candidate agent is capable of binding to the Toso receptor.

In a preferred embodiment, the methods comprise differential screeningto identity bioactive agents that are capable of modulating theactivitity of the Toso receptors. In this embodiment, the methodscomprise combining a Toso cell surface receptor and a competitor in afirst sample. A second sample comprises a candidate bioactive agent, acell surface Toso receptor and a competitor. The binding of thecompetitor is determined for both samples, and a change, or differencein binding between the two samples indicates the presence of an agentcapable of binding to the Toso receptor and potentially modulating itsactivity. That is, if the binding of the competitor is different in thesecond sample relative to the first sample, the agent is capable ofbinding to the Toso receptor.

Alternatively, a preferred embodiment utilizes differential screening toidentify drug candidates that bind to the native Toso receptor, butcannot bind to modified receptors. The structure of the Toso receptormay be modeled, and used in rational drug design to synthesize agentsthat interact with that site. Drug candidates that modulate apoptosisare also identified by screening drugs for the ability to either enhanceor reduce the apoptotic response which is triggered by binding to theToso receptor.

Positive controls and negative controls may be used in the assays.Preferably all control and test samples are performed in at leasttriplicate to obtain statistically significant results. Incubation ofall samples is for a time sufficient for the binding of the agent to theToso receptor. Following incubation, all samples are washed free ofnon-specifically bound material and the amount of bound, generallylabeled agent determined. For example, where a radiolabel is employed,the samples may be counted in a scintillation counter to determine theamount of bound compound.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g., albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Alsoreagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

Screening for agents that modulate the activity of Toso may also bedone. In a preferred embodiment, methods for screening for a bioactiveagent capable of modulating the activity of Toso comprise the steps ofadding a candidate bioactive agent to a sample of Toso, as above, anddetermining an alteration in the biological activity of Toso.“Modulating the activity of Toso” includes an increase in activity, adecrease in activity, or a change in the type or kind of activitypresent. Thus, in this embodiment, the candidate agent should both bindto Toso (although this may not be necessary), and alter its biologicalor biochemical activity, as defmed herein. The methods include both invitro screening methods, as are generally outlined above, and in vivoscreening of cells for alterations in the presence, distribution,activity or amount of Toso.

Thus, in this embodiment, the methods comprise combining a Toso sampleand a candidate bioactive agent, and testing the Toso biologicalactivity as is known in the art to evaluate the effect of the agent onthe activity of Toso. By “Toso activity” or grammatical equivalentsherein is meant the ability of Toso after activation to modulateapoptosis. As outlined herein, upon T cell activation, Toso isactivated, initiating a signalling pathway that results in modulation ofapoptosis. Such modulation may result in response to either of theextracellular or cytoplasmic domains of Toso and may correspond to adecrease or an increase in apoptosis. In a preferred embodiment, theactivity of the extracellular or cytoplasmic domain of Toso isincreased; in another preferred embodiment, the activity of theextracellular or cytoplasmic domain of Toso is decreased. Thus,bioactive agents that are antagonists (i.e. decrease the activity ofToso proteins) are preferred in some embodiments, and bioactive agentsthat are agonists (i.e., increase the activity of Toso proteins) may bepreferred in other embodiments. For example, agents which bind to a Tosoreceptor, but do not allow activation or signalling of the receptorscould be antagonists. In addition, agents which bind to a Toso receptor,may increase activation or signalling of the receptors, and thus act asagonists.

In a preferred embodiment, the invention provides methods for screeningfor bioactive agents capable of modulating the activity of a Tosocell-surface receptor. The methods comprise adding a candidate bioactiveagent, as defined above, to a cell comprising Toso cell-surfacereceptors or the Toso cytoplasmic domain. Preferred cell types include,but are not limited to mammalian cells, for example T cells such asJurkat cells, 293 or 31 cells. The cells contain a recombinant nucleicacid that encodes a Toso receptor; that is, the cells express Tosoeither at the surface of the cell or within the cell. In a preferredembodiment, a library of candidate agents are tested on a plurality ofcells.

The cells are then exposed to an apoptotic agent that will induceapoptosis in control cells, i.e., cells of the same type but that do notcontain the exogeneous nucleic acid encoding Toso. Suitable apoptoticagents include, but are not limited to, Fas-mediated apoptosis inducers,including the Fas ligand (FasL) and anti-Fas receptor antibodies(particularly monoclonal antibodies), chemotherapeutic agents, forexample, cisplatin, taxol, methotrexate, etc.; tumor necrosisfactor-alpha (TNF-α); FADD, PMA; ionomycin; and staurosporine.

The effect of the candidate agent on apoptosis is then evaluated. IfToso is acting, i.e., there is no antagonistic agent present, the cellswill not undergo programmed cell death. However, if antagonistic agentsare present, the cells will undergo apoptosis.

Detection of apoptosis may be done as will be appreciated by those inthe art. In one embodiment, annexin is used. Annexin will stain cellsundergoing apoptosis. Accordingly, annexin can be used as an affinityligand, and attached to a solid support such as a bead, a surface, etc.and used to pull out apoptotic cells. Similarly, annexin can be used asthe basis of a fluorescent-activated cell sorting (FACS) separation.Apoptosis may also be detected by staining of cells with propidiumiodide, by use of mitochondrial dyes, or by use of FRET constructs.

In this way, bioactive agents are identified. Compounds withpharmacological activity are able to enhance or interfere with theactivity of the Toso receptor. The compounds having the desiredpharmacological activity may be administered in a physiologicallyacceptable carrier to a host, as previously described. The agents may beadministered in a variety of ways, orally, parenterally e.g.,subcutaneously, intraperitoneally, intravascularly, etc. Depending uponthe manner of introduction, the compounds may be formulated in a varietyof ways. The concentration of therapeutically active compound in theformulation may vary from about 0.1-100 wt. %.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

Without being bound by theory, it appears that Toso is an importantsignalling step in apoptosis. Accordingly, disorders based on mutant orvariant Toso genes may be determined. In one embodiment, the inventionprovides methods for identifying cells containing variant Toso genescomprising determining all or part of the sequence of at least oneendogenous Toso genes in a cell. As will be appreciated by those in theart, this may be done using any number of sequencing techniques. In apreferred embodiment, the invention provides methods of identifying theToso genotype of an individual comprising determining all or part of thesequence of at least one Toso gene of the individual. This is generallydone in at least one tissue of the individual, and may include theevaluation of a number of tissues or different samples of the sametissue. For example, putatively cancerous tissue of an individual or anydiseased tissue are preferred samples. The method may include comparingthe sequence of the sequenced Toso gene to a known Toso gene, i.e., awild-type gene.

The sequence of all or part of the Toso gene can then be compared to thesequence of a known Toso gene to determine if any differences exist.This can be done using any number of known homology programs, such asBestfit, etc. In a preferred embodiment, the presence of a a differencein the sequence between the Toso gene of the patient and the known Tosogene is indicative of a disease state or a propensity for a diseasestate, as outlined herein.

The present discovery relating to the role of Toso in apoptosis thusprovides methods for inducing apoptosis in cells. In a preferredembodiment, the Toso proteins, and particularly Toso fragments, areuseful in the study or treatment of conditions which are mediated byapoptosis, i.e. to diagnose, treat or prevent apoptosis-mediateddisorders. Thus, “apoptosis mediated disorders” or “disease state”include conditions involving immune disorders or cellular processesmediated by apoptosis, as well as conditions which have inappropriateapoptosis or a lack thereof. Accordingly, apoptosis mediated disordersinclude, but are not limited to, any disease characterized by lymphoidor T cell overactivity, including, but not limited to Sjogrens, mixedconnective tissue disease, autoimmune disorders including, but notlimited to, lupus (SLE), rheumatoid arthritis (RA), multiple sclerosis,and autoimmune diseases which are tissue specific, for example liver(hepatitis), kidney (nephritis) or Hashimotois (thyroiditis); diseaseswhere T cells actively destroy cells, for example, cytotoxic effectsincluding, but not limited to, transplant rejection, disease conditionsbased on graft vs. host or host vs. graft reactions; conditions wherecells of any kind that are not dying express Toso appropriately, forexample, cancer of T or B cell origin (where increased apoptosis wouldbe desirable), including but not limited to, leukemias and lymphomas, orChrohn's disease, skin inflammatory disorders (psoriasis, eczema); anddiseases secondary to altered immunoglobulin production such asWaldenstroms, and multiple myeloma.

Thus, in one embodiment, methods of modulating apoptosis in cells ororganisms are provided. In one embodiment, the methods compriseadministering to a cell an anti-Toso antibody that reduces or eliminatesthe biological activity of the endogenous Toso receptor. Alternatively,the methods comprise administering to a cell or organism a recombinantnucleic acid encoding a Toso receptor. As will be appreciated by thosein the art, this may be accomplished in any number of ways. In apreferred embodiment, the activity of Toso is increased by increasingthe amount of Toso in the cell, for example by overexpressing theendogenous Toso or by administering a gene encoding Toso, using knowngene-therapy techniques, for example. In a preferred embodiment, thegene therapy techniques include the incorporation of the exogeneous geneusing enhanced homologous recombination (EHR), for example as describedin PCT/US93/03868, hereby incorporated by reference in its entireity.

In one embodiment, the invention provides methods for diagnosing anapoptosis related condition in an individual. The methods comprisemeasuring the activity and expression of Toso in a tissue from theindividual or patient, which may include a measurement of the amount orspecific activity of Toso. This activity is quantified and compared tothe activity of Toso from either an unaffected second individual or froman unaffected tissue from the first individual. When these activitiesare different, the first individual may be at risk for an apoptosismediated disorder. In this way, for example, monitoring ofimmunosuppression may be done, by monitoring the levels of Toso.Similarly, Toso levels may correlate to levels of T cell activity orlevels of immune responsiveness.

In one embodiment, the Toso proteins of the present invention may beused to generate polyclonal and monoclonal antibodies to theextracellular or cytoplasmic domains of Toso proteins, which are usefulas described herein. Similarly, the Toso proteins can be coupled, usingstandard technology, to affinity chromatography columns. These columnsmay then be used to purify Toso antibodies. In a preferred embodiment,the antibodies are generated to epitopes unique to the Toso protein;that is, the antibodies show little or no cross-reactivity to otherproteins. These antibodies find use in a number of applications. Forexample, the Toso antibodies may be coupled to standard affinitychromatography columns and used to purify Toso proteins. The antibodiesmay also be used as blocking polypeptides, as outlined above, since theywill specifically bind to the Toso protein.

In one embodiment, a therapeutically effective dose of a Toso isadministered to a patient. By “therapeutically effective dose” herein ismeant a dose that produces the effects for which it is administered. Theexact dose will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques. As isknown in the art, adjustments for Toso degradation, systemic versuslocalized delivery, and rate of new protease synthesis, as well as theage, body weight, general health, sex, diet, time of administration,drug interaction and the severity of the condition may be necessary, andwill be ascertainable with routine experimentation by those skilled inthe art.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and organisms. Thus themethods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

The administration of the Toso proteins of the present invention can bedone in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds and inflammation, the Toso may be directly applied as a solutionor spray.

The pharmaceutical compositions of the present invention comprise a Tosoprotein in a form suitable for administration to a patient. In thepreferred embodiment, the pharmaceutical compositions are in a watersoluble form, such as being present as pharmaceutically acceptablesalts, which is meant to include both acid and base addition salts.“Pharmaceutically acceptable acid addition salt” refers to those saltsthat retain the biological effectiveness of the free bases and that arenot biologically or otherwise undesirable, formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as acetic acid,propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid,malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike. “Pharmaceutically acceptable base addition salts” include thosederived from inorganic bases such as sodium, potassium, lithium,ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminumsalts and the like. Particularly preferred are the ammonium, potassium,sodium, calcium, and magnesium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are incorporated by reference.

EXAMPLE I

Molecular Cloning and Chromosomal Localization of Toso.

Jurkat cells (human T cell line) were infected with a retroviral JurkatT cell cDNA library to screen for cDNAs that encode inhibitory moleculesfor Fas-induced apoptosis. A retroviral library containing 2×10⁶independent cDNA inserts was constructed from Jurkat cell mRNA bystandard methods (Kinoshita and Nolan, unpublished) using a retrovirusvector pBabeMN (Kinoshita, et al. (1997)). The library was transfectedinto an ecotropic virus packaging cell line, ΦNX-Ampho, as describedpreviously. Jurkat cells were spin-infected with the supernatant fromΦNX-A cells resulting in 20-40% infection using this method asdetermined by doping of the library with a marker retroviruspBabeMN-LacZ or pBabeMN-Lyt-2-α (194 amino acids), which does not havecytoplasmic domain (Tagawa, et al., Proc. Natl. Acad. Sci, 83:3422-3426(1986)). Jurkat cells were aliquoted into 96-well plates in mediacontaining 10 ng/ml of anti-human Fas mAb, CH11, (Kamiya BiomedicalCompany, California 91359, U.S.A.) for 15 days. Jurkat cells, underconditions empirically derived, were sensitive to Fas-mediated apoptosiswith a spontaneous survival rate under our conditions of 2-3 per 10⁶cells. Cells that survived the Fas-mediated killing were identified byoutgrowth in the 96 well plate format, expanded, total RNA extracted,and cDNA inserts rescued using RT-PCR (AMV reverse transcriptase fromPromega, Wis. 53711, U.S.A. and Vent DNA polymerase from New EnglandBiolabs, Inc., Massachusetts 01915, U.S.A.) with primers 5′-GCT CAC TTACAG GCT CTC TA (SEQ ID NO:22) (LibS) and 5′-CAG GTG GGG TCT TTC ATT CC(SEQ ID NO:23) (LibA), which were located 282 bp and 56 bp nucleotidesupstream and downstream of cDNA insert cloning sites. After an initialdenaturation at 94° C. for 5 minutes, each cycle of amplificationconsisted of 30 second denaturation at 94° C., followed by a 30second-annealing at 58° C. and 2 minutes extension at 72° C. After 35cycles, the final product was extended for 10 minutes at 72° C. Therescued inserts were digested with BamHI-Sall (Promega) or BstXI(Promega), and ligated into the pBabeMN retrovirus vector. The clonedretrovirus containing the novel insert was infected into Jurkat cells.Cells were cultured with 10 ng/ml anti-Fas mAb to confirm whether theinhibitory effect was caused by cDNA inserts of retrovirus. 26 cloneswere obtained that were resistant to Fas-induced apoptosis, of which 12carried cDNA inserts. After a second round of anti-Fas screening, oneclone, termed here Toso, demonstrated potent inhibition of Fas-inducedapoptotic signaling.

The cDNA insert of Toso was found to contain a 5′-non-coding region of73 nucleotides, a coding region of 1173 nucleotides (390 amino acids)and a 3′-non-coding region of 665 nucleotides. (See FIG. 1, SEQ IDNO:1). The ATG initiation codon is contained within a standard Kozakconsensus sequence. Kyte-Doolittle hydropathy plot analysis showed thatToso has two hydrophobic regions: the amino-terminal residues from 1 to17 correspond to the deduced signal sequence (underlined) and residuesfrom 254 to 272 (double underlined) correspond to a presumptivetransmembrane region [Hofmann and Stoffel, f993, analysis was performedusing DNAsis-Mac V2.0 (Hitachi Software Engineering, Co. Ltd., Japan)],suggesting that Toso is a type I integral membrane protein. (See FIG.2b). The predicted molecular weight of Toso is 41 kDa. The cytoplasmicregion of Toso has a basic amino acid-rich region (from R²⁷⁴ to R³²³), aproline-rich region (from P³³⁴ to P³⁴⁶), and an acidic amino acid-richregion (from E³⁷⁸ to D³⁸⁴) (See FIGS. 2a and 2 b, SEQ ID NO:2). BLASTsearch analysis revealed that Toso is a unique gene (Altschul, et al.,(1990)). The extracellular domain of Toso has homology to theimmunoglobulin variable (IgV) domains, which is characterized by motifsin the β-strand B, D and F regions, (residues VTLTC (SEQ ID NO:24),RV(or F or I) and DSG(or A)-Y-CA) (SEQ ID NO:25)) (Williams and Barclay,Ann. Rev. Immunol., 6:381-405 (1988)). Importantly, the cysteines in theIgV-like motif VTIKC (SEQ ID NO:26) at position 33 in Toso, as well asthe cysteine in the IgV-like motif DSGVYAC (SEQ ID NO:27) at position98, are appropriately distanced as in other IgV-like domains to form adisulphide bond. Toso also contains within the Ig domains two additionalcysteines that are not conserved in other IgV-like domains. Thus, thepresumptive extracellular domain has all the requisite features thatdemarcate it as a potential IgV-like domain. The cytoplasmic region ofToso has partial homology to FAST kinase, acid sphingomyelinase, insulinreceptor substrate-1 (IRS-1) and the apoptosis inhibitor from Orgyiapseudotsugata nuclear polyhedrosis virus (Op-IAP) (FIG. 3), which mightfunction to initiate some of the signaling systems acted upon by Toso.

Poly (A)⁺ RNA was prepared from Jurkat cells stimulated for 24 hourswith 10 ng/ml PMA (SIGMA) and 500 ng/ml lonomycin (SIGMA). The firststrand of cDNA was synthesized with 10 μg Poly (A)⁺ RNA using oligo-dTprimers and performed PCR with primers, 5′-AGA ATT CTC TCT AGG GGC TCTTGG ATG (SEQ ID NO:28) (See FIG. 1 (SEQ ID NO:1) where the EcoRI site isunderlined) and 5′-ATA AAG CTT CTC AGG GCA CAG ATA GAT GG (SEQ ID NO:29)(HindIII site is underlined), which were located 23 bp and 136 bpnucleotides upstream and downstream of the Toso coding region,respectively. The 1.3 kbp fragment was ligated into pBluescript SK(+).Five independent clones were picked up and sequenced using cyclesequencing ready reaction kit (Perkin Elmer). The deduced amino-acidsequences from the five independent clones were completely identical tothe gene from the cDNA library screening, although two silent mutationswere found within the original gene as compared to the PCR consensussequences.

The Toso gene was mapped to a human chromosome by using a panel of 17human X Chinese hamster hybrid cell lines derived from severalindependent fusion experiments (Francke et al., 1986). PCR primers usedto amplify Toso sequence derived from the 3′ untranslated region were5′-AGA GGC ATA GCT ATT GTC TCG G (SEQ ID NO: 30) (sense; located 369 bpdownstream of the coding region), and 5′-ACA TTT GGA TCA GGG CAA AG (SEQID NO:31) (anti-sense; 508 bp downstream of the coding region). The sizeof the PCR product was 159 bp. The PCR conditions were 94° C., 90seconds; then 35 cycles of 94° C., 20 seconds; 55° C., 30 seconds; 72°C., 45 seconds; followed by 72° C., 5 minutes. Specific PCR productswere obtained from human genomic DNA, and hybrid cell lines that carryhuman chromosome 1. The PCR product was sequenced to confirm itsidentity.

To map the Toso gene locus more precisely, two human radiation hybrid(RH) mapping panels were typed by PCR. GeneBridge 4 (WhiteheadInstitute/MIT Genome Center) and Stanford G3 (Stanford Human GenomeCenter), were obtained from Research Genetics, Inc. (Cox, et al.,Science, 250:245-250 (1990); Walter, et al., Net Genet, 7:22-28 (1994)),and samples were typed using the primers and PCR conditions describedabove. Results of the maximum likelihood analysis (Boehnke, et al., Am.J. Hum. Genet., 49:1174-1188 (1991)) were obtained by submitting the rawscores to: http://www-genome.wi.mit.edu/cgibin/contig/rhmapper.p1 andhttp://wwwshgc.stanford.edu/rhserver2/rhserver_form.htm1. Thecytological localization of the Toso gene was deduced from thecytogenetic information about the flanking markers in Bray-Ward et al(Bray-Ward, et al., Genomics, 32:1-14 (1996)). In the Stanford G3mapping panel, Toso cosegregated with chromosome 1 marker D1S3553 on all83 Stanford G3 panel RH cell lines. D1 S3553 is a known marker ofchromosome 1 bin 115 on the SHGC RH map. In the GeneBridge 4 mappingpanel, Toso is located 5.4 cR₃₀₀₀ and 1.7 cR₃₀₀₀ from D1 S504 andW1-9641, respectively. The order of loci in this region from centromereto qter is: D1S412- D1S306 D1S504-Toso-W1-9641-D1S491-D1S237. Accordingto Bray-Ward et al. (1996), the YACs containing the more proximalmarkers D1S412 (bin 104), D1S477 (bin 109) and D1S504 (bin 114) weremapped to 1q25-q32, 1q31-q32 and 1q25-q32 respectively, and the YACscontaining the more distal markers D1 S491 (bin 118), D1 S414 (bin 121)and D1 S237 (bin 124) were mapped to essentially the same region,1q31-q32, 1q31-q32 and 1q32-q41, respectively. Thus, the Toso gene islocated at 1q31-q32, a region in which several chromosomal abnormalitiesrelating to leukemias are localized.

Toso is a negative regulator of Fas-mediated cell death in lymphoidcells, and may therefore be involved in oncogenic events or resistanceto chemotherapy (Friesen, et al., Nature Medicine, 2:574-577 (1996)).The gene for Toso localizes within human chromosome region 1q31-q32.Chromosomal changes in 1q32 are frequently observed in human cancer,including various types of hematopoietic malignancies and solid tumors(Jinnai, et al., Am. J. Hematol, 35:118-124 (1990); Mertens, et al.,Cancer Res., 57:2765-2780 (1997); Mitelman, et al., Nat. Genet., 417-474(1997); Schmid and Kohler, Cancer Genet. Cytogenet, 11:121-23 (1984);Shah, et al., Cancer Genet. Cytogenet, 61:183-192 (1992); Waghray, etal., Cancer Genet. Cytogenet, 23:225-237 (1986); Yip, et al., CancerGenet. Cytogenet, 51:235-238 (1991)). Furthermore, studies in nude micedemonstrated that duplication of the chromosome segment of 1 q11-q32 isassociated with proliferation and metastasis of human chroniclymphocytic leukemic B-cells (Ghose, et al., Cancer Res., 50:3737-3742(1990)), suggesting the presence of dominantly acting growth regulatoryor cell survival genes. Thus, Toso is a candidate for evaluation as aproto-oncogene in several proliferative and metastatic neoplasms.

EXAMPLE 2

Toso Inhibits Fas-, TNFα- and FADD-Induced Apoptosis.

Jurkat cells that express the receptor for ecotropic murine retroviruses(“Jurkat.ecoR”) were infected with retroviruses that express Toso andcontrol vectors, pBabeMN-Toso, pBabeMN-lacZ and pBabeMN-Lyt-2-α′ (α′form of mouse CD8α chain) (Tagawa, et al. (1986)). Jurkat.ecoR cellswere infected with pBabeMN-lacZ, pBabeMN-Lyt-2-α′, and pBabeMN-Toso. At72 hours postinfection, infection frequency of pBabeMN-lacZ andpBabeMN-Lyt-2α′ were determined to be 45% and 58%, respectively. Jurkatcells were then cultured with 10 ng/ml anti-Fas mAb for 24 hours. After12 or 24 hours, the cells were stained with 100 μg/ml ethidium bromide(SIGMA) and 100 μg/ml acridine orange (SIGMA). Apoptotic cells andnon-apoptotic cells were identified with UV microscopy as described(MacGahon, et al., The End of the (Cell) Line: Methods for the Study ofApoptosis in vitro, in Methods in cell biology, L. J. Schwartz and B. A.Osborne, eds., San Diego, Calif., Academic Press, Inc., pp. 172-173(1995)).

Jurkat.ecoR cells expressing Toso were resistant to apoptosis induced by10 ng/ml of anti-Fas mAb, whereas Jurkat cells, Jurkat.ecoR cells andJurkat.ecoR cells that expressed lacZ or Lyt-2-α′, all succumbed toapoptotic death (FIG. 4a).

Staurosporine is a bacterial alkaloid that is a broad spectrum inhibitorof protein kineses (Tamaoki and Nakano, Biotechnology, 8:732-735 (1990))and induces programmed cell death in various cell lines and dissociatedprimary cells in culture (Ishizaki, et al., J. Cell Biol., 121:899-908(1993); Jacobson, et al., Nature, 361:365-369 (1993); Raff, et al.,Science, 262:695-700 (1993)). Ceramide generation is implicated in asignal transduction pathway that mediates programmed cell death inducedby Fas and TNF-α (Cifone, et al., J. Exp. Med., 180:1547-1552 (1994);Obeid, et al., Science, 259:1769-1771 (1993)). pBabeMN-LacZ infectedcells were counted by microscopic observation; infection frequency wasdetermined to be 57%. At 72 hours postinfection, Jurkat.ecoR cells andJurkat.ecoR cells infected with pBabeMN-lacZ and pBabeMN-Toso werecultured with anti-Fas mAb, staurosporine and ceramide for 24 hours.Although Jurkat.ecoR cells expressing Toso were resistant toFas-mediated apoptosis over a range of antiFas dilutions, these cellswere not resistant to any concentration of staurosporine- orceramide-induced apoptosis (FIG. 4b).

The Fas receptor has homology to the TNF-α receptor, and these tworeceptors share analogous signaling systems as well as severalintracellular mediators (Hsu, et al., Cell, 84:299-308 (1996)). Theprotective effect of Toso against TNF-α-induced apoptosis was tested byculturing Jurkat.ecoR cells expressing Lyt-2-α′ or Toso with 10 ng/ml ofanti-Fas mAb or 1 μg/ml of TNF-α in the presence of 0.1 μg/ml ofcyclohexamide (CHX) for 12 hours and apoptotic cells were counted. Theinfection frequency of pBabeMN-Lyt-2-α′ was determined to be 58%. Tosoinhibited Fas induced apoptosis in the presence of CHX and alsoprotected against TNF-α-induced apoptosis in comparison to Jurkat.ecoRexpressing Lyt-2-α′ (FIG. 4d). Thus the TNF-α and Fas signaling pathwaysmay converge at a common point that can be inhibited by Toso.

Fas-mediated apoptosis is activated through FADD. For FADD-inducedapoptosis, mouse FADD (a gift from Dr. Angeles Estelles, Dept. Mol.Pharm., Stanford Univ.) was ligated into pBabeMN retroviral vector.Jurkat.ecoR cells expressing Lyt-2-α′ or Toso were infected withpBabeMN-LacZ or pBabeMN-FADD. After 24 hours infection with FADD, thecells were stained with ethidium bromide and acridine orange and countedthe apoptotic cells. The effect of Toso on FADD-induced apoptosis wasinvestigated by infecting Jurkat.ecoR cells expressing Lyt-2-α′ or Toso,with pBabeMN-LacZ or pBabeMN-FADD. The reinfection efficiency wasapproximately 40% using pBabeMN-LacZ. Jurkat.ecoR cells were infectedwith pBabeMN-Lyt-2-α′, and pBabeMN-Toso. Infection frequency ofpBabeMN-Lyt-2-α′ was determined to be 72%. Jurkat.ecoR cells expressingLyt-2-α′ or Toso were infected with pBabeMNLacZ or pBabeMN-FADD andapoptotic cells were counted at 24 hours postinfection. Infectionfrequency of pBabeMN-lacZ in Jurkat.ecoR cells expressing Lyt-2-α′ andToso was determined to be 39% and 43%, respectively. As shown in FIG.4c, FADD induced apoptosis in 45% of control Jurkat cells. However, FADDfailed to induce apoptosis in Jurkat.ecoR cells constitutivelyexpressing Toso. The results also suggest that Toso's effect is not dueto down regulation of FADD gene expression.

The downstream effects of Toso on known inhibitors of apoptosis, wereevaluated by western blot analysis of Bc1-2 and BCI XL expression levelsin Toso expressing cells. Bc1-2 overexpression can block Fas-inducedapoptosis as well as staurosporine-induced apoptosis (data not shown).No change in the levels of expression of Bc1-2 or BcI XL was observed byWestern blot (data not shown). Thus, it appears that intracellularsignaling events generated by FADD can be directly and efficientlyblocked by signals emanating from Toso at a point prior to engagement ofBc1-2 and Bc1 XL.

The effect of overexpression of Toso on processing of caspase-8, whichassociates with FADD, was evaluated. The processed form (p20) of FLICEafter Fas activation was greatly reduced in pBabeMN-Toso-infectedJurkat.ecoR cells in comparison with control Jurkat.ecoR cells (see FIG.5a). To detect caspase-8, whole-cell lysates (2×10⁶ cells per lane) wereresolved by SDS-PAGE, transferred to an membrane and processed with goatanti-Mch5 p20 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif. 95060, U.S.A.) as described above. This data indicates that Tosoinhibits caspase-8 processing after Fas activation.

Recently several groups have reported cFLIP is a caspase-8 inhibitor. Weperformed semi-quantitative RT-PCR to detect cFLIP mRNA expression. Todetect cFLIP mRNA expression, a 1.1 kbp fragment (998-2061) of the cFLIPgene (U97074) was amplified with primers 5′-GGG AGA AGT AAA GAA CAA AG(SEQ ID NO:32) and 5′-CGT AGG CAC AAT CAC AGC AT (SEQ ID NO:33) for 35cycles as described above. The sequence of the 1.1 kbp PCR product wasverified using cycle sequencing ready reaction kit (Perkin Elmer, Calif.94404, U.S.A.). As a control, β-actin cDNA was amplified for 15 and 25cycles as described above. cFLIP expression was induced by Toso (FIG.5b). These results strongly suggest that the extracellular domain ofToso inhibits Fas-induced apoptosis by preventing caspase-8 processingthrough cFLIP upregulation.

Toso did not inhibit staurosporine-induced programmed cell death andstaurosporine has been shown to activate caspase-8 (Jacobsen, et al., J.Cell Biol., 133:1041-1051 (1996)). Therefore, additional Toso effects donot occur downstream, nor at the level, of caspase-8. Supporting this,Toso also did not inhibit ceramide-induced apoptosis, which actsdownstream or independent of caspase-8 as demonstrated in experimentsusing the caspase-8-specific inhibitor peptide, DEVD-CHO (Gamen, et al.,FEBS Lett., 390:232-237 (1996)), which does not inhibit ceramide-inducedapoptosis. Overexpression of Bc1-2 or Bc1-XL is known to preventapoptosis in response to ceramide and staurosporine (Geley, et al., FEBSLett., 400:15-18 (1997); Susin, et al., J. Exp. Med., 186:25-37 (1997);Takayama, et al., Cell, 80:279-284 (1995); Zhang, et al., Proc. Natl.Acad. Sci. USA, 93:5325-5328 (1996)). Toso did not change the expressionlevels of Bc1-2 nor Bc1-XL in Jurkat cells, showing that neither Bc1-2nor Bc1-XL were involved in the protective activities of Toso. Takentogether then, Toso activates an inhibitory pathway that preventscaspase-8 activation following Fas stimulation through upregulation ofcFLIP, and not by blocking apoptotic signals downstream or at the levelof caspase-8. This explains the apparent specificity of the blockade toTNF family-related surface receptors that use caspase-8 for apoptoticsignaling.

Cells expressing Toso alone were mixed with an equal number of cellsexpressing lacZ. After one round of Fas stimulation, no lacZ-expressingcells remained as assayed by X-gal. In addition, Jurkat.ecoR cells wereinfected with pBabeMN-Toso-IRES-GFP. After infection, cells werecultured with (α-Fas (+)) or without (α-Fas (−)) 50 ng/ml of anti-FasmAb. In the absence of anti-Fas mAb treatment (Fas (−))., 46% GFPnegative cells and 54% GFP positive cells were observed inpBabeMN-Toso-IRES-GFP-infected Jurkat.ecoR cells. After five daysculture with anti-Fas mAb, survivors were obtained frompBabeMN-Toso-1RES-GFP-infected Jurkat.ecoR cells, but not from controlpBabeMN-IRES-GFP-infected Jurkat.ecoR cells (data not shown); 99.7% ofsurviving Jurkat cells expressed GFP as shown in FIG. 4e (Fas(+)). Thesedata indicate that cells that express the extracellular domain of Tosoare protected from Fas-induced apoptosis and suggests that Toso does notexert its effect as a secreted form.

EXAMPLE 3

The Immunoglobulin Domain and the Transmembrane Region of Toso AreRequired for Inhibition of Fas-induced Apoptosis.

The C-terminus deletion mutants (TosoΔ(377-390).HA, TosoΔ(334-390).HA,TosoΔ(281-390).HA and TosoA(252-390).HA), the N-terminus deletion mutant(TosoΔ(29-187).HA) and the fusion protein (Lyt-2/Toso(271-390).HA) ofthe extracellular domain and transmembrane region from Lyt-2-α′ and thecytoplasmic domain from Toso, which have the influenza virushemmagglutinin tag (HA) in C-terminus, were generated by. Primers in theantisense orientation, carrying the 20 nucleotide sequences of Tosolocated upstream of the deletion sites, HA tag sequence and an in-frametermination codon, as well as NcoI site, were synthesized. The DNAfragment of the Toso gene from the XhoI site located in theextracellular domain to the Ncol site that is located in 3′ non-codingregion was replaced with the PCR products amplified from pBabeMN-Tosousing LibS and each primer described above. A primer forTosoΔ(29-187).HA in the antisense orientation carrying the 20nucleotides located after the leader peptides of Toso and XhoI site wassynthesized. The DNA fragment from DraIII site, which is located 190 bpupstream of cDNA insert cloning sites, to XhoI site in pBabeMN-Toso.HAwas replaced with the PCR product amplified from pBabeMN-Toso using LibSand the primer. For Lyt2/Toso(271-390).HA, primer in sense orientationwhich is carried a BamHI site and the 20 nucleotides located upstream ofthe cytoplasmic domain was synthesized. The DNA fragment from Bcl Isite, which is located in the end of transmembrane region of Lyt-2-α′,to SalI site, which is located downstream of Lyt-2-α′ cloning sites inpBabeMN-Lyt-2-α′, was replaced with the PCR product amplified frompBabeMN-Toso.HA using LibA and the primer. All mutants generated by PCRwere verified by DNA sequencing using cycle sequencing ready reactionkit. Toso deletion mutants prepared as described above wereepitope-tagged in order to delineate the regions responsible foranti-apoptotic signal transduction, (FIGS. 6a and 6 b). Toso.HA (fusedto the hemagglutinin, HA, tag) had an apparent molecular weight of 60kDa, suggesting Toso is heavily glycosylated. The cell surfaceexpression of Fas using anti-human Fas mAb, CH11, was determined by FACSto explore whether Toso has an effect on Fas expression. Fas wasexpressed at similar levels on the surface of cells expressing eitherfull-length Toso, Toso deletion mutants, or control vector. Thus, theextracellular domain of Toso neither downregulates Fas, nor directlyinterferes with the ability of the antibody to bind and presumablystimulate Fas.

Jurkat.ecoR cells were infected with pBabeMN-Lyt-2-α′. HA, pBabeMN-Toso.HA, pBabeMN-TosoΔ(377-390). HA, pBabeMN-TosoΔ(334-390). HA,pBabeMN-TosoΔ(281-390).HA, pBabeMNTosoΔ(252-390).HA andpBabeMN-TosoΔ(29-187).HA. Jurkat cells were cultured with 10 ng/mlanti-Fas mAb for 24 hours and apoptotic cells were counted. Apoptosiswas readily induced in control Jurkat.ecoR cells and Jurkat.ecoR cellsexpressing Lyt-2-α′.HA, whereas apoptosis was markedly inhibited inJurkat.ecoR cells that expressed Toso.HA (FIG. 6a). Deletions of regionsof the cytoplasmic domain of Toso from 334 to 390 still inhibitedapoptosis. Moreover, a deletion of Toso lacking the entire cytoplasmicdomain still retained substantial anti-apoptotic ability. Thus, thecytoplasmic domain of Toso is not absolutely required for theanti-apoptotic effects on Fas antibody-stimulated cells. (See Example 5,below) These results indicate that the homologies observed in thecytoplasmic region of Toso, as shown in FIG. 3, are not the only sourcesof the anti-apoptotic signals generated by a Toso complex, although thecytoplasmic regions are required for enhancing the anti-apoptoticeffects of Toso.

The Toso mutant lacking the transmembrane and cytoplasmic domainsdemonstrated that inhibition of Fas-induced apoptosis by Toso requiresits insertion into membranes. As shown in FIG. 6a, solubleTosoΔ(252-390).HA afforded no protection from apoptosis. Expression ofthe TosoΔ(252-390).HA protein was confirmed by western blot analysis ofculture supernatants. Supernatants derived from pBabeMN-TosoA(252-390).HA-transfected 293T cells did not inhibit Fas-induced apoptosis,indicating that a membrane-proximal event dependent on cis-localizationof Toso is required for blockade of the Fas-mediated death signal.

Many cell surface receptor complexes act through oligomerization andmost immunoglobulin (Ig) domain proteins exist in homodimeric andheterodimeric Ig forms, functioning as self-assembling systems.Disruption of the Ig domain of Toso completely abrogated theanti-apoptotic ability of Toso (TosoΔ(29-187).HA). (See FIG. 6a).Further, a chimeric Lyt-2Toso fusion protein in which the cytoplasmicdomain of Toso was coupled to the extracellular and transmembrane regionof Lyt-2-α′ (α′ form of murine CD8α, which forms homodimers at the cellsurface) (Tagawa, et al., (1986)) failed to inhibit Fas-inducedapoptosis. Furthermore, anti-mouse CD8a mAb (Lyt-2) was used tocrosslink the Lyt-2-Toso chimeras and induce multimerization of the Tosocytoplasmic domains. These results suggest that some form of Ig domainmediated dimerization of Toso is required to initiate the anti-apoptoticeffect in conjunction with the cytoplasmic region of Toso or other cellsurface Toso-associating proteins. Toso.HA-expressing Jurkat.ecoR cells(5×10⁶ cells) were incubated with 2 mM BS3 (PIERCE, Rockford, Ill.61105, U.S.A.) for 1 hour at 4° C. After incubation, 1M Tris-HCI wasadded to a final concentration of 10 mM and cells were incubated for 15minutes at 4° C. Whole-cell lysates were resolved by SDS PAGE,transferred to a membrane and processed with mouse monoclonalantihemagglutinin antibody (HA.11) (Babco) as described above. Apparentcrosslinking molecular complexes at 150, 240, 300 kDa were detected (SeeFIG. 6c). This result first indicates that Toso is a surface expressedreceptor. The results are consistent with an association of Toso withanother surface protein(s) of molecular weight 90 kDa. The severalmolecular weights observed for the crosslinked complexes are alsominimally consistent with stochiometric mixtures of 60 and 90 kDamolecules.

Deletion analysis of Toso indicated that surface expression of theimmunoglobulin V-like region is necessary to inhibitFas-induced-apoptosis and that the cytoplasmic domain of Toso isinsufficient and indeed partly expendable for the anti-apoptoticfinction. Deletion of the cytoplasmic domain resulted in abrogation ofonly about half of the anti-apoptotic effect. This suggests that Tosomust be expressed at the cell surface in a manner where it presumablyinteracts other surface molecule(s) that propagate an anti-apoptoticsignal. Most immunoglobulin family receptors are homo- or heterodimersthat can become activated through ligand interactions. Crosslinkingexperiments revealed multiple potential higher-order complexes (150,240, and 300 kDa), suggesting at least one partner of 90 kDa thatinteract with Toso. We suspect that Toso forms a heterodimer with thisother surface protein to collaborate in initiating the anti-apoptoticsignal that leads to cFLIP induction. Interactions of surface-expressedToso complexes with ligands on or near target cells might also modulatethe ability of Toso to provide anti-apoptotic signaling. We arecurrently investigating the existence of such ligands and contributorymolecules.

A model summarizing the results is shown in FIG. 10. In this model,stimulation through of the T cell receptor complex transmits activationsignals leading to upregulation of Fas and FasL. Activation also inducesToso expression, providing the potential for anti-apoptotic signals thatprotect against Fas-mediated apoptosis. Toso accomplishes this byforming homo- or heterodimers at the cell surface to generate signalsthat inhibit the initiation or propagation of caspase-8 activation bycFLIP. It is also possible that Toso requires an extracellular ligandthat might modulate its activities. The signaling pathway activated byToso is clearly important as it leads to induced expression of cFLIP(Irmler, et al. (1997); Srinivasula, et al. (1997)).

EXAMPLE 4

T Cell Signaling Leading to Apoptosis is Blocked by Activated Toso.

Poly (A)⁺ RNA was prepared from Jurkat cells or Jurkat cells stimulatedfor 24 hours with 10 ng/ml of phorbol 12-myristate 13-acetate (PMA;SIGMA Chemical Company, Missouri 63178, U.S.A.) and 1 μg/mlphytohemagglutinin (PHA; SIGMA) or 10 ng/ml PMA and 500 ng/ml lonomycin(SIGMA). Poly (A)⁺ RNA (5 μg) was subjected to electrophoresis through1% agarose gel containing 2.2 M formaldehyde, and transferred to HybondN⁺ membrane (Amersham Life Science Inc., Illinois 60005, U.S.A.).Hybridization was carried out according to the manufacturer'srecommendation. A specific probe for the Toso coding region (1.2 kbp)was synthesized with PCR from pBabeMN-Toso using 5′-AGG GGC TCT TGG ATGGAC (SEQ ID NO:34) (TosoS) and 5′-CTG GGG TTG GGG ATA GC (SEQ ID NO:35)(TosoA). As a control probe, the human β-actin cDNA control probe(CLONTECH Laboratories, Inc., California 94303-4230) was used. Probeswere labeled with ³²p using a random-primed labeling kit, Prime-a-Gene(Promega). Human RNA Master Blot and Human Immune System Multiple TissueNorthern Blot II (CLONTECH Laboratories) were used to survey Toso mRNAexpression in several human tissues. Toso expression was observed inlymph nodes, lung and kidney. In addition to these tissues, we detectedfaint signals from spleen, thymus, liver, heart and salivary gland.Tissues which were analyzed for Toso mRNA include: A1: Whole brain, A2:Amygdala, A3: Caudate nucleus, A4: Cerebellum, A5: Cerebral cortex, A6:Frontal lobe, A7: Hippocampus, A8: Medulla oblongata, B1: Occipitallobe, B2: Putarnen, B3: Substantia nigra, B4: Temporal lobe, B5:Thalamus, B6: Subthalamic nucleus, B7: Spinal cord, C1: Heart, C2:Aorta, C3: Skeletal muscle, C4: Colon, C5: Bladder, C6: Uterus, C7:Prostate, C8: Stomach, D1: Testis, D2: Ovary, D3: Pancreas, D4:Pituitary gland, D5: Adrenal gland, D6: Thyroid gland, D7: Salivarygland, D8: Mammary gland, E1: Kidney, E2: Liver, E3: Small intestine,E4: Spleen, E5: Thymus, E6: Peripheral leukocyte, E7: Lymph node, E8:Bone marrow, F1: Appendix, F2: Lung, F3: Trachea, F4: Placenta, G1:Fetal brain, G2: Fetal heart, G3: Fetal kidney, G4: Fetal liver, G5:Fetal spleen, G6: Fetal thymus, G7: Fetal lung. (FIG. 7a). Using HumanImmune System Multiple Tissue Northern Blot II, and film exposed at −70°C. with an intensifying screen for one day, endogenous Toso mRNA speciesof 2.0 (major), 2.8, 3.5 and 4.3 kbp were detected in lymph node andspleen (see FIG. 7b). The nucleotide length of the cDNA was 1.9 kbp,suggesting that the additional bands might either be alternative spliceproducts or incompletely processed messages. Toso expression was alsoobserved in peripheral blood leukocytes, thymus (FIG. 7b). Expression inbone marrow and fetal liver was much lower than that in lymph node andspleen, as seen after overexposure of the blot (data not shown).

The expression of Toso in several human cell lines was analyzed bysemi-quantitative RT-PCR involving amplification of the 1.2 kbp-codingregion of Toso (FIG. 7c). The first strand of cDNA was synthesized with10 μg total RNA from several human cell lines and peripheral bloodmononuclear cells. PCR was performed for 35 cycles using TosoS andTosoA. After an initial denaturation at 94° C. for 5 minutes, each cycleof amplification consisted of 30 second denaturation at 94° C., followedby a 30 second-annealing at 58° C. and 2 minutes extension at 72° C.After 35 cycles, the final product was extended for 10 minutes at 72° C.PCR products were electrophoresed through 1.0% agarose gel andtransferred to Hybond N+membrane. The BamHI-Xhol fragment (510 bp) ofthe Toso-coding region were labeled with ³²p. Hybridization was carriedout as described above.

Toso mRNA was detected in lymphoid cell lines such as Jurkat cells (Tcell leukemia), CemT4 cells (T cell leukemia), MolT-4 cells (T cellleukemia), HB11.19 cells (B cell lymphoma), a kind gift from Dr. Cleary,M. L., Stanford Univ., and Reh cells (acute lymphocytic leukemia; non T;non B, ATCC). HL-60 cells (promyelocytic leukemia, ATCC) displayed aconsistently weak signal. In contrast, Toso PCR products were notdetected in non-hematopoietic cell lines including HepG2 cells(hepatoblastoma, a kind gift from Dr. Blau, Stanford Univ.), 293 cells(kidney; transformed with adenovirus, ATCC) and Hela cells (cervix;adenocarcinoma, ATCC). Toso therefore is constitutively expressed incells of hematopoietic cells.

Toso was expressed in several human cell lines including Jurkat cells,CemT4 cells (human T cell leukemia), SupTI cells (human T cell leukemia,a kind gift from Dr. Cleary, M. L., Stanford Univ.), Oli-Ly8 cells(human B cell line; transformed with EBV), AMK cells (human B cell line;transformed with EBV), both a gift from Dr. Negrin, R. S., StanfordUniv., Reh cells (acute Iymphocytic leukemia; non T; non B), HL-60 cells(promyelocytic leukemia) and HepG2 cells (hepatoma) using pBabeMN-TosoIRES neo to allow cotranslational selection with Geneticin (GIBCO BRL).All of the human T cell lines and one of the human B cell lines, Ocl-Ly8cells, in which Toso was overexpressed, were inhibited for apoptosisinduced by anti-Fas mAb, whereas no significant protection was observedagainst Fas-induced apoptosis in the other cell lines (data not shown).Thus, the anti-apoptotic effect of Toso also is limited to certainclasses of hematopoietic cells, suggesting the presence oftissue-specific mediators in these cells.

T cell activation results in increased expression of Fas and FasL on thecell surface. This is paradoxical, as it is clear that T cells do notkill themselves after such induction, whereas overexpression of Fas andFasL in other cell types does lead to cell death. In vitro, PMA andlonomycin can induce apoptosis in T cells (Oyalzu, et al., Biochem.Biophys. Res. Commun., 213:994-1001 (1995)) by mimicking certain aspectsof CD3 engagement, including upregulation of Fas and FasL. One functionof Toso might be to inhibit T cell activated self-killing and that thelevels of Toso might become increased following T cell activation,helping to render Jurkat cells partially resistant to upregulated Fasand FasL. Expression of Toso mRNA in Jurkat cells was examined bynorthern hybridization. As shown in FIG. 8a, an endogenous Toso mRNAspecies of 2.8 kbp was detected in resting Jurkat cells, althoughexpression was seen after overexposure of the blot (data not shown).Toso mRNA expression increased, including minor species (2.0, 3.5, 4.3,5.5 kbp), after stimulation of Jurkat cells with PMA and PHA (15-foldincrease) or PMA in combination with lonomycin (25-fold increase). Thus,Toso can be induced following T-cell activation. We hypothesize thatinduced Toso expression would correlate with resistance to Fas-mediatedapoptosis.

Jurkat.ecoR cells, Jurkat.ecoR cells infected with pBabeMN-lacZ, andpBabeMN-Toso-infected clones were precultured with 10 ng/ml PMA and 500ng/ml lonomycin for 12 hours and then incubated with 10 ng/ml ofanti-Fas mAb for 24 hours, and as shown in FIG. 8b, Jurkat cells weresusceptible to anti-Fas mAb-induced apoptosis as well asPMA/lonomycin-induced apoptosis. However, following activation withPMA/lonomycin one third of Jurkat cells were clearly resistant toanti-Fas mAb induced apoptosis. These results suggest that Jurkat cellsactivate a protective system that blocks Fas-mediated apoptosis,supporting the contention that induced Toso is a mediator in thisprotective effect.

We further tested whether Toso expression could rescueactivation-induced programmed cell death. We randomly picked fivepBabeMN-Toso-infected Fas resistant Jurkat T cell clones and used theseto assay the inhibitory effect of Toso on PMA/lonomycin-inducedapoptosis. All five clones exhibited significant resistance toPMA/lonomycin-induced apoptosis, as well as continued strong resistanceto Fas-induced apoptosis (FIG. 8c). Control clones displayed theexpected killing effect when activated with PMA and lonomycin. Toso notonly inhibited apoptosis activated by Fas and TNF-α, but also inhibitedapoptosis induced by certain classes of T cell activation events.

Normal T cells at early stages of activation are resistant toFas-induced apoptosis but become Fas sensitive at late stages ofactivation (Klas, et al., (1993)). Toso expression kinetics inperipheral blood mononuclear cells were examined after PHA stimulationusing by semi-quantitative RT-PCR. Peripheral blood leucocyte (PBL) fromhealthy volunteers were isolated by Histopaque-1077 (Sigma) densitycentrifugation. Adherent cells were removed by adherence to plasticculture vessels. Cells were activated with phytohemagglutinin (PHA)-P (1μg/ml) for 24 hours washed, and cultured with 20 U/ml of recombinanthuman IL-2 (R&D Systems Inc., Minneapolis, Minn. 55413, U.S.A.). Cellswere cultured for one to seven days (day 1, 3, 5, and 7). Tosoexpression was observed at day 1 and upregulated expression at day 3after activation. However, Toso expression was clearly decreased at days5 (FIG. 9a), correlating with Fas sensitivity studies (Klas, et al.(1993)). To perform mixed lymphocyte culture, PBL were treated with 20μg/ml of mitomycin-C (stimulating cells, SC) for 3 hours and washed. SCwere adjusted to 7×10⁵ cells/ml and cultured with an equal volume andcell density of PBL (responding cells, RC) from another donor (Clot, etal., Immunology, 29:445-453 (1975)). Further, allogenic stimulation inmixed lymphocyte cultures was performed to determine whether Toso isactivated in primary immune cells upon T cell activation. As shown inFIG. 9b, Toso expression was also rapidly induced in the presence ofstimulator cells on day 1; however Toso expression in mixed lymphocytecultures was reduced by day 6 to levels even lower than seen on day 1and responder cells alone at day 6. These results further confirm asupportive role for Toso induced resistance to Fas-mediated death duringT lymphocyte activation.

Natural T cell resistance to Fas-induced apoptosis shows atime-dependent kinetics (Klas, et al. (1993)). By day 6 post-activation,T cells become susceptible to Fas-induced death. In addition, activationof Jurkat cells by PMA/lonomycin induces a significant increase in Fasligand expression which is thought to promote apoptosis (Oyalzu, et al.(1995); Brunner, et al., Nature, 373:441-444 (1995)). However,PMA/lonomycin-activated Jurkat cells were not as efficiently induced toundergo apoptosis by anti-Fas mAb treatment compared to unstimulatedJurkat cells (FIG. 8b). This suggested that Jurkat cells become at leastpartly resistant to anti-Fas mAb-induced apoptosis after T cellsignaling, mimicking processes observed in natural T cells. mRNAexpression of Toso in Jurkat cells, as well as in peripheral T cells, isstrongly upregulated upon stimulation with T cell activators. Further,overexpression of Toso protected Jurkat cells against PMA/lonomycin-induced apoptosis.

This is consistent with the proposal that Toso expression, whichtransiently increased and then decreased in peripheral blood mononuclearcells after activation with PHA or allogenic stimulation, is responsiblefor the temporary Fas resistance in T cells. Hence, the results areconsistent with the hypothesis that Toso may be involved inactivation-induced resistance to apoptosis of T cells during an immuneresponse. We conclude from the results that the inhibitory effect of theextracellular domain of Toso in activation-induced apoptosis isattributable to the inhibition of Fas-mediated signal transductionthrough inhibition of caspase-8 by c-FLIP induction.

The finding that Toso can exert cell-specific and signaling pathwayspecific effects on apoptosis suggests that other polypeptides existthat act upon the Fas death induction cascade. Critically, the fact thatsignalling by the extracellular domain of Toso induces expression ofcFLIP suggests the existence of a regulatable transcription cascade thatcan be activated to block Fas-mediated apoptosis in some cell types. Asshown here, high efficiency gene transduction using a retroviralapproach, like other cDNA cloning approaches (Vito, et al., Science,271:521-525 (1996); Kitamura, et al., Prac. Natl. Acad. Sci.,92:9146-9150 (1995)), allows functional cloning of genes with highthroughput and accuracy. Further analysis of the Toso pathway coupledwith gene disruption analysis in animals will further clarify theoverall role that the extracellular domain of Toso plays in modulatingactivation-induced T-cell apoptosis in vivo.

EXAMPLE 5

The Cytoplasmic Domain of Toso Promotes Cell Death in Murine pre-BCells.

70Z/3 cells were incubated with virus at 32° C. for 12 hours includinginitial spinning and achieved 70-80 % infection efficiency estimatedusing FACS analysis for pBabeMN-Lyt-2α. 70 Z3 cells kept about 80%viability at the end of 12 hours incubation with virus. However, afterinfection, we observed rapid cell death (about 70% of cells were dead)in 70Z/3 cells infected with pBabeMN-Toso, not in 70 Z3 cells withpBabeMN- Lyt-2α nor with supernatant of ΦNX-E cells (FIG. 11).Supernatant from pBabeMN-Toso transfected 293T cells, which is theparental cell line of ΦNX-E and ΦNX-A cells, did not induce rapid celldeath to 70Z/3 cells. These results suggest that gene products of Tosoinduced rapid cell death. Most dead cells after infection showedapoptotic nuclei under microscopic observation, suggesting Toso inducedapoptosis to 70Z/3 cells.

To clarify which region was responsible for apoptotic signaltransduction, a set of deletion mutants of the Toso cDNA was prepared asshown in Example 3. The mutated Toso cDNA was ligated into pBabeMNretroviral vector and infected 70Z/3 cells. As shown in Table A, below,massive cell death was observed in 70Z/3 cells infected withpBabeMN-Toso.HA, -TosoΔ(377-390).HA, -TosoΔ(334-390). HA andLyt-2/Toso(271-390).HA, but not pBabeMN-TosoΔ(252-390).HA andpBabeMN-TosoΔ(29-187).HA. Full length Lyt-2 did not induce rapid celldeath to 70Z/3 cells after infection (data not shown).Lyt-2/Toso(271-390).HA. was most effective in promoting cell death in70Z/3 cells, suggesting that the cytoplasmic region was responsible formassive cell death in 70 Z3 cells.

The Toso-induced cell death in 70Z/3 cells, suggests that Toso works notonly for protection against Fas-induced apoptosis but also for promotionof cell death. The cytoplasmic domain from A²⁸¹ to A³³³ is responsiblefor promotion of cell death. BLAST search reveals that this region haspartial homology to FAST kinase and acid sphingomyelinase which isinvolved in Fas-induced apoptosis. When the cytoplasmic domain of Tosois compared to the “death domain” from several molecules, thecytoplasmic domain of Toso did not show any homology to known “deathdomain”, including the consensus sequence as described. The promotion ofcell death by Toso was not observed in several cell lines. Cell deathinduced by Toso may be observed in some stages of B cell development.

Table A indicates the effect of Toso deletion mutants on promotion ofapoptosis. 70Z/3 cells were infected with pBabeMN-Lyt-2α.HA,pBabeMN-Toso.HA, pBabeMN-TosoΔ(377-390).HA, pBabeMN-TosoΔ(334-390).HA,pBabeMN-TosoΔ(281-390).HA, pBabeMN-TosoΔ(252-390).HA andpBabeMN-TosoΔ(29-187).HA. After infection, the stained cells wereincubated with phosphate-buffered saline including 100 μg/ml of ethidiumbromide (SIGMA) and 100 μg/ml of acridine orange (SIGMA). Viable cellswere identified with UV microscopy. The percentage of viable cells isexpressed as mean±SD of triplicate cultures.

Infected-Virus Encoding % Viable Cells Lyt-2.HA 73 ± 5 4.8.HA 30 ± 44.8Δ(377-390).HA 31 ± 5 4.8Δ(334-390).HA 29 ± 2 4.8Δ(281-390).HA 75 ± 24.8Δ(252-390).HA 78 ± 3 4.8Δ(29-187).HA 83 ± 3 Lyt-2/4.8(271-390).HA  5± 3

33 1 1910 DNA Homo sapiens 1 aaaggagtaa gcagcgtgtc tccatccccc tctctaggggctcttggatg gaccttgcac 60 tctagaaggg acaatggact tctggctttg gccactttacttcctgccag tatcaggggc 120 cctgaggatc ctcccagaag taaaggtaga gggggagctgggcggatcag ttaccatcaa 180 atgcccactt cctgaaatgc atgtgaggat atatctgtgccgggagatgg ctggatctgg 240 aacatgtggt accgtggtat ccaccaccaa cttcatcaaggcagaataca agggccgagt 300 tactctgaag caatacccac gcaagaatct gttcctagtggaggtaacac agctgacaga 360 aagtgacagc ggagtctatg cctgcggagc gggcatgaacacagaccggg gaaagaccca 420 gaaagtcacc ctgaatgtcc acagtgaata cgagccatcatgggaagagc agccaatgcc 480 tgagactcca aaatggtttc atctgcccta tttgttccagatgcctgcat atgccagttc 540 ttccaaattc gtaaccagag ttaccacacc agctcaaaggggcaaggtcc ctccagttca 600 ccactcctcc cccaccaccc aaatcaccca ccgccctcgagtgtccagag catcttcagt 660 agcaggtgac aagccccgaa ccttcctgcc atccactacagcctcaaaaa tctcagctct 720 ggaggggctg ctcaagcccc agacgcccag ctacaaccaccacaccaggc tgcacaggca 780 gagagcactg gactatggct cacagtctgg gagggaaggccaaggatttc acatcctgat 840 cccgaccatc ctgggccttt tcctgctggc acttctggggctggtggtga aaagggccgt 900 tgaaaggagg aaagccctct ccaggcgggc ccgccgactggccgtgagga tgcgcgccct 960 ggagagctcc cagaggcccc gcgggtcgcc gcgaccgcgctcccaaaaca acatctacag 1020 cgcctgcccg cggcgcgctc tggagcggac gctgcaggcacaggggaggc ccccgttccc 1080 ggccccggag cgccgttgcc ccccgccccg ctgcaggtgtctgaatctcc ctggctccat 1140 gccccatctc tgaagaccag ctgtgaatac gtgagcctctaccaccagcc tgccgccatg 1200 atggaggaca gtgattcaga tgactacatc aatgttcctgcctgacaact ccccagctat 1260 cccccaaccc caggctcgga ctgtggtgcc aaggagtctcatctatctgc tgatgtccaa 1320 tacctgcttc atgtgttctc agagccctca tcacttcccatgccccatct cgactcccat 1380 ccccatctat ctgtggccct gagcatggct ctgcccccaggtcgtcttgc acaccttggc 1440 agccccctgt agttgacagg taagctgtag gcatgtagagcaattgtccc aatgccactt 1500 gcttcctttc caagccgtcg aacagactgt gggatttgcagagtgtttct tccatgtctt 1560 tgaccacagg gtgttgttgc tgccaggctc tagatcacatggcatcaggc tggggcagag 1620 gcatagctat tgtctcgggc atccttccca gggttgggtcttacacaaat agaaggctct 1680 tgctctgagt tatgtgacgt gcctcagccc catggactaagcaggggtct ggtataaaca 1740 ctcctggaaa cgcctttgcc ctgatccaaa tgttagcacttgctagtgaa cgtctactta 1800 tctcaagttc tatgctaaag gcaatttatc ttgatgtgatgataaaccaa acttattagc 1860 aagatatgca tatatatcca taaattctct ttactctgtctccatccttt 1910 2 390 PRT Homo sapiens 2 Met Asp Arg Trp Leu Trp Pro LeuTyr Phe Leu Pro Val Ser Gly Ala 1 5 10 15 Leu Arg Ile Leu Pro Glu ValLys Val Glu Gly Glu Leu Gly Gly Ser 20 25 30 Val Thr Ile Lys Cys Pro LeuPro Glu Met His Val Arg Ile Tyr Leu 35 40 45 Cys Arg Glu Met Ala Gly SerGly Thr Cys Gly Thr Val Val Ser Thr 50 55 60 Thr Asn Phe Ile Lys Ala GluTyr Lys Gly Arg Val Thr Leu Lys Gln 65 70 75 80 Tyr Pro Arg Lys Asn LeuPhe Leu Val Glu Val Thr Gln Leu Thr Glu 85 90 95 Ser Asp Ser Gly Val TyrAla Cys Gly Ala Gly Met Asn Thr Asp Arg 100 105 110 Gly Lys Thr Gln LysVal Thr Leu Asn Val His Ser Glu Tyr Glu Pro 115 120 125 Ser Trp Glu GluGln Pro Met Pro Glu Thr Pro Lys Trp Phe His Leu 130 135 140 Pro Tyr LeuPhe Gln Met Pro Ala Tyr Ala Ser Ser Ser Lys Phe Val 145 150 155 160 ThrArg Val Thr Thr Pro Ala Gln Arg Gly Lys Val Pro Pro Val His 165 170 175His Ser Ser Pro Thr Thr Gln Ile Thr His Arg Pro Arg Val Ser Arg 180 185190 Ala Ser Ser Val Ala Gly Asp Lys Pro Arg Thr Phe Leu Pro Ser Thr 195200 205 Thr Ala Ser Lys Ile Ser Ala Leu Glu Gly Leu Leu Lys Pro Gln Thr210 215 220 Pro Ser Tyr Asn His His Thr Arg Leu His Arg Gln Arg Ala LeuAsp 225 230 235 240 Tyr Gly Ser Gln Ser Gly Arg Glu Gly Gln Gly Phe HisIle Leu Ile 245 250 255 Pro Thr Ile Leu Gly Leu Phe Leu Leu Ala Leu LeuGly Leu Val Val 260 265 270 Lys Arg Ala Val Glu Arg Arg Lys Ala Leu SerArg Arg Ala Arg Arg 275 280 285 Leu Ala Val Arg Met Arg Ala Leu Glu SerSer Gln Arg Pro Arg Gly 290 295 300 Ser Pro Arg Pro Arg Ser Gln Asn AsnIle Tyr Ser Ala Cys Pro Arg 305 310 315 320 Arg Ala Arg Gly Ala Asp AlaAla Gly Thr Gly Glu Ala Pro Val Pro 325 330 335 Gly Pro Gly Ala Pro LeuPro Pro Ala Pro Leu Gln Val Ser Glu Ser 340 345 350 Pro Trp Leu His AlaPro Ser Leu Lys Thr Ser Cys Glu Tyr Val Ser 355 360 365 Leu Tyr His GlnPro Ala Ala Met Met Glu Asp Ser Asp Ser Asp Asp 370 375 380 Tyr Ile AsnVal Pro Ala 385 390 3 73 PRT Homo sapiens 3 Val Thr Ile Lys Cys Pro LeuPro Glu Met His Val Arg Ile Tyr Leu 1 5 10 15 Cys Arg Glu Met Ala GlySer Gly Thr Cys Gly Thr Val Val Ser Thr 20 25 30 Thr Asn Phe Ile Lys AlaGlu Trp Lys Gly Arg Val Thr Leu Lys Gln 35 40 45 Tyr Pro Arg Lys Asn LeuPhe Leu Val Glu Val Thr Gln Leu Thr Glu 50 55 60 Ser Asp Ser Gly Val TyrAla Cys Gly 65 70 4 79 PRT Homo sapiens 4 Leu Ser Leu Thr Cys Thr ValSer Gly Ser Thr Phe Ser Asn Asp Tyr 1 5 10 15 Tyr Thr Trp Val Arg GlnPro Pro Gly Arg Gly Leu Glu Trp Ile Gly 20 25 30 Tyr Val Phe Tyr His GlyThr Ser Asp Asp Thr Thr Pro Leu Arg Ser 35 40 45 Arg Val Thr Met Leu ValAsp Thr Ser Lys Asn Gln Phe Ser Leu Arg 50 55 60 Leu Ser Ser Val Thr AlaAla Asp Thr Ala Val Tyr Tyr Cys Ala 65 70 75 5 73 PRT Homo sapiens 5 ValThr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn 1 5 10 15Tyr Ala Asn Trp Val Gln Gln Lys Pro Asp His Leu Phe Thr Gly Ile 20 25 30Gly Gly Thr Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe Ser Gly 35 40 45Ser Leu Ile Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala Gln Thr 50 55 60Glu Asp Glu Ala Ile Tyr Phe Cys Ala 65 70 6 72 PRT Homo sapiens 6 ThrSer Leu Asn Cys Thr Phe Ser Asp Ser Ala Ser Gln Tyr Phe Trp 1 5 10 15Trp Tyr Arg Gln His Ser Gly Lys Ala Pro Lys Ala Leu Met Ser Ile 20 25 30Phe Ser Asn Gly Glu Lys Glu Glu Gly Arg Phe Thr Ile His Leu Asn 35 40 45Lys Ala Ser Leu His Phe Ser Leu His Ile Arg Asp Ser Gln Pro Ser 50 55 60Asp Ser Ala Leu Tyr Leu Cys Ala 65 70 7 75 PRT Homo sapiens 7 Val ThrLeu Arg Cys Lys Pro Ile Ser Gly His Asn Ser Leu Phe Trp 1 5 10 15 TyrArg Gln Thr Met Met Arg Gly Leu Glu Leu Leu Ile Tyr Phe Asn 20 25 30 AsnAsn Val Pro Ile Asp Asp Ser Gly Met Pro Glu Asp Arg Phe Ser 35 40 45 AlaLys Met Pro Asn Ala Ser Phe Ser Thr Leu Lys Ile Gln Pro Ser 50 55 60 GluPro Arg Asp Ser Ala Val Tyr Phe Cys Ala 65 70 75 8 74 PRT Homo sapiens 8Val Glu Leu Thr Cys Thr Ala Ser Gln Lys Lys Ser Ile Gln Phe His 1 5 1015 Trp Lys Asn Ser Asn Gln Ile Lys Ile Leu Gly Asn Gln Gly Ser Phe 20 2530 Leu Thr Lys Gly Pro Ser Lys Leu Asn Asp Arg Ala Asp Ser Arg Arg 35 4045 Ser Leu Trp Asp Gln Gly Asn Phe Pro Leu Ile Ile Lys Asn Leu Lys 50 5560 Ile Glu Asp Ser Asp Thr Tyr Ile Cys Glu 65 70 9 80 PRT Homo sapiens 9Ala Lys Met Ser Cys Glu Ala Lys Thr Phe Pro Lys Gly Thr Thr Ile 1 5 1015 Tyr Trp Leu Arg Glu Leu Gln Asp Ser Asn Lys Asn Lys His Phe Glu 20 2530 Phe Leu Ala Ser Arg Thr Ser Thr Lys Gly Ile Lys Tyr Gly Glu Arg 35 4045 Val Lys Lys Asn Met Thr Leu Ser Phe Asn Ser Thr Leu Pro Phe Leu 50 5560 Lys Ile Met Asp Val Lys Pro Glu Asp Ser Gly Phe Tyr Phe Cys Ala 65 7075 80 10 76 PRT Homo sapiens 10 Val Thr Ile Thr Cys Pro Phe Thr Tyr AlaThr Arg Gln Leu Lys Lys 1 5 10 15 Ser Phe Tyr Lys Val Glu Asp Gly GluLeu Val Leu Ile Ile Asp Ser 20 25 30 Ser Ser Lys Glu Ala Lys Asp Pro ArgTyr Lys Gly Arg Ile Thr Leu 35 40 45 Gln Ile Gln Ser Thr Thr Ala Lys GluPhe Thr Val Thr Leu Lys His 50 55 60 Leu Gln Leu Asn Asp Ala Gly Gln TyrVal Cys Gln 65 70 75 11 84 PRT Homo sapiens SITE (6)..(82) The aminoacids at positions at 6, 7, 9-18, 20, 22, 25-32, 34, 35, 37-48, 50, 51,54-65, 71, 73-76, 80, and 82 can be any amino acid. 11 Val Thr Leu ThrCys Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa PheXaa Trp Xaa Arg Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Leu Xaa XaaTyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Tyr Xaa XaaArg Phe Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Phe SerLeu Thr Ile Xaa Asn Xaa Xaa Xaa Xaa Asp Ser Ala Xaa 65 70 75 80 Tyr XaaCys Ala 12 43 PRT Homo sapiens 12 Gln Arg Pro Arg Gly Ser Pro Arg ProArg Ser Gln Asn Asn Ile Tyr 1 5 10 15 Ser Ala Cys Pro Arg Arg Ala ArgGly Ala Asp Ala Ala Gly Thr Gly 20 25 30 Glu Ala Pro Val Pro Gly Pro GlyAla Pro Leu 35 40 13 35 PRT Homo sapiens 13 Arg Arg Pro Arg Gly Glu ProGly Pro Arg Ala Pro Arg Pro Thr Glu 1 5 10 15 Gly Ala Thr Cys Ala GlyPro Gly Glu Ser Trp Ser Pro Ser Pro Asn 20 25 30 Ser Met Leu 35 14 36PRT Homo sapiens 14 Met Pro Pro Arg Tyr Gly Ser Leu Arg Gln Ser Cys ProArg Ser Gly 1 5 10 15 Arg Glu Gln Gly Gln Asp Gly Thr Ala Gly Ala ProGly Leu Leu Trp 20 25 30 Met Gly Leu Val 35 15 19 PRT Homo sapiens 15Glu Ser Pro Trp Leu His Ala Pro Ser Leu Lys Thr Ser Cys Glu Tyr 1 5 1015 Val Ser Leu 16 19 PRT Homo sapiens 16 Asp Ala Pro Trp Gln Gln His AlaArg Trp Tyr Asp Arg Cys Glu Tyr 1 5 10 15 Val Leu Leu 17 19 PRT Homosapiens 17 Gln Gln Pro Leu Leu His Pro Pro Glu Pro Lys Ser Pro Gly GluTyr 1 5 10 15 Val Asn Ile 18 19 PRT Homo sapiens 18 Trp Glu Pro Trp LeuPro Ala Glu Ala Leu Thr Arg Leu Arg Ile Gly 1 5 10 15 Gly Phe Tyr 19 19PRT Homo sapiens 19 Gln Pro Ala Ala Met Met Glu Asp Ser Asp Ser Asp AspTyr Ile Asn 1 5 10 15 Val Pro Ala 20 19 PRT Homo sapiens 20 Thr Glu AlaCys Val Val Arg Asp Ala Asp Asn Glu Pro His Ile Glu 1 5 10 15 Arg ProAla 21 19 PRT Homo sapiens 21 Gln Pro Ala Pro Arg Glu Glu Glu Thr GlyThr Glu Glu Tyr Met Lys 1 5 10 15 Met Asp Leu 22 20 DNA Homo sapiens 22gctcacttac aggctctcta 20 23 20 DNA Homo sapiens 23 caggtggggt ctttcattcc20 24 5 PRT Homo sapiens 24 Val Thr Ile Lys Cys 1 5 25 7 PRT Homosapiens 25 Asp Ser Gly Val Tyr Ala Cys 1 5 26 27 DNA Homo sapiens 26agaattctct ctaggggctc ttggatg 27 27 29 DNA Homo sapiens 27 ataaagcttctcagggcaca gatagatgg 29 28 22 DNA Homo sapiens 28 agaggcatag ctattgtctcgg 22 29 20 DNA Homo sapiens 29 acatttggat cagggcaaag 20 30 20 DNA Homosapiens 30 gggagaagta aagaacaaag 20 31 20 DNA Homo sapiens 31 cgtaggcacaatcacagcat 20 32 18 DNA Homo sapiens 32 aggggctctt ggatggac 18 33 17 DNAHomo sapiens 33 ctggggttgg ggatagc 17

I claim:
 1. A method for screening for a bioactive agent capable of modulating the activity of a Toso cell-surface receptor, said method comprising the steps of: a) adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding a Toso cell-surface receptor, wherein said recombinant nucleic acid will hybridize under high stringency conditions to the nucleic acid sequence depicted in FIG. 1 (SEQ ID NO:1) or its complement; 0 b) exposing said cell to an apoptotic agent that will induce apoptosis; and c) determining the effect of the candidate bioactive agent on apoptosis.
 2. A method according to claim 1, wherein a library of candidate bioactive agents is added to a plurality of cells comprising a recombinant nucleic acid encoding a Toso cell-surface receptor.
 3. A method according to claim 1 further comprising adding a labeling agent that will label apoptotic cells.
 4. A method according to claim 3 further comprising separating apoptotic cells from non-apoptotic cells.
 5. A method according to claim 4 wherein said separation is done by FACS.
 6. A method according to claim 3 wherein said labeling agent is annexin.
 7. A method according to claim 1 wherein said apoptotic agent is selected from the group consisting of an anti-Fas antibody, TNF-α, FADD, cycloheximide, PMA, ionomycin and chemotherapeutic agents.
 8. A method according to claim 1, wherein said recombinant nucleic acid comprises the coding region of the nucleic acid sequence depicted in FIG. 1 (SEQ ID NO:1).
 9. A method according to claim 1, wherein said Toso protein comprises the amino acid sequence depicted in FIG. 2A (SEQ ID NO:2). 